WO2009149166A2 - Methods and compositions for the diagnosis and treatment of proliferative disorders - Google Patents

Abstract

The present invention identifies expression markers associated with neurofibromatosis 1 (NF1) by examining gene expression in tissue from lesions from neurofibromas, plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The present invention also provides for diagnostic markers as well as markers that can be used to monitor disease states, disease progression, drug toxicity, drug efficacy and drug metabolism. The present invention relates to novel, rapid, reliable and effective assays for screening and identifying pharmaceutically effective compounds that act as a therapeutic agent for the treatment of a proliferative disorder. In another aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene related to the NF1-Ras pathway.

Description

Methods And Compositions For The Diagnosis And Treatment Of Proliferative Disorders

[0001] This invention was made with government support under grant KOl-

NS049191-01A1, awarded by the National Institute of Neurological Disorders and Stroke and under grant DAMD W81XWH-04- 1-0273, awarded by the Department of Defense. The government has certain rights in the invention.

Cross-Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial

No. 61/058,428, entitled "Methods and Compositions for the Diagnosis and Treatment of Proliferative Disorders," filed on June 3, 2008, herein incorporated by reference in its entirety for all purposes.

Background of the Invention

[0003] A genetic defect underlies neurofibromatosis type 1 (NFl), which is inherited as an autosomal dominant trait and affects 1 :3000 humans (Rasmussen and Friedman, 2000). Analysis of progressive changes downstream of NFl mutation has been complicated by the wide spectrum of clinical manifestations in NFl patients and by the diversity of cell types involved. The hallmark of NFl is the development of peripheral nerve sheath tumors. At least 95% of NFl patients have multiple dermal and cutaneous neurofibromas (Friedman and Birch, 1997), benign tumors that typically appear in adolescence and may cause significant morbidity to the patients. Approximately 30% of NFl patients develop plexiform neurofibromas, which are larger and can occur congenitally. Questions as fundamental as whether there are molecular differences between dermal and plexiform neurofibroma are to date unanswered. Differences between the types of neurofibroma are implied as a plexiform neurofibroma may transform to a malignant peripheral nerve sheath tumor (MPNST), a life threatening sarcoma (Evans et al., 2002; Zoller et al., 1997).

[0004] Additional approaches are needed to complement current serological assays in defining clinically important sub-types of NFl -related disorder with respect to disease behavior and progression.

[0005] In particular, current biomarkers have not been efficacious in distinguishing among the various subtypes of NFl -related disorders. Accordingly, there is a need for biomarkers that can form the basis for diagnostic tools that can effectively classify, characterize and predict the severity of NFl -related disorder.

[0006] There is further the need for biomarkers that can be used to predict disease progression. The instant invention satisfies these needs and provides related advantages as well.

Brief Summary of the Invention

[0007] The present invention identifies the global changes in gene expression associated with neurofibromatosis 1 (NFl) by examining gene expression in tissue from lesions from neurofibromas, plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The present invention also identifies expression profiles which serve as useful diagnostic markers as well as markers that can be used to monitor disease states, disease progression, drug toxicity, drug efficacy and drug metabolism.

[0008] The present invention provides for gene expression patterns that distinguish human Neurofibromatosis Type 1 (NFl)-derived rumor and cell line samples from normal primary human Schwann cell cultures. The NFl samples include: malignant peripheral nerve sheath tumors (MPNST), primary benign neurofibromas, and purified, primary genetically defined neurofibroma Schwann cells.

[0009] The present invention relates to novel, rapid, reliable and effective assays for screening and identifying pharmaceutically effective compounds that act as a therapeutic agent for the treatment of a proliferative disorder. The phrase "cellular characteristic associated with a proliferative disorder" as used herein is intended to include any feature or property, whether biological or biochemical, of a cell or cellular population that is indicative of a proliferative disorder, particularly that of NFl or an NFl related disease. For example, the characteristic may be but is not limited to, migration, proliferation, rate of cell growth, or cellular adhesion. The cellular characteristic may be that of individual cells or a population of cells. [0010] The present invention relates to biological markers and methods of using biological markers for the diagnosis and prognosis of NFl -related disorders. The present invention further relates to biological markers that may be used to predict and characterize disease behavior in individuals having an NFl -related disorder.

[0011] The present invention identifies gene clusters which act as biomarkers and therapeutic targets in tumors.

[0012] The present invention further relates to biological markers that may be used to predict disease progression, particularly neurofibroma progression disease.

[0013] In a related aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene in Tables 1-3.

[0014] In another aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene related to the NFl-Ras pathway.

[0015] In another aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene involving Eyes Absent (EYA), Dachshund (DACH), and Sine Oculis (SIX).

[0016] The above summary of the present invention is not intended to describe each embodiment or every implementation of the present invention. Advantages and attainments, together with a more complete understanding of the invention, will become apparent and appreciated by referring to the following detailed description and claims. All publications mentioned herein are incorporated herein by reference in their entirety for all purposes, which are described in the publications which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such a disclosure by virtue of prior invention.

Detailed Description of the Invention

[0018] Definitions

[0019] For convenience, certain terms employed in the specification, examples and claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[0020] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] The phrase "alteration" as used herein is intended to encompass any mutation or deletion of a gene, including truncation, deletion of the entire sequence or a portion of the gene, or one or more mutations that result in ablated or significantly attenuated gene function, such that the net result of the alteration is to essentially or substantially reduce the function of a gene of interest such that the assay as described herein can be effectively carried out to identify potential therapeutic agent. The term may also encompass any mutation that results in suppression or altered translation or transcription of the gene of interest, such that the gene function is essentially or substantially reduced in function. Determination of alterations with respect to a particular gene that satisfies the above- definition requires only routine experimentation and is well within the ordinary skill in the art.

[0022] "Association" or "associated with" in context of the present invention means that the nucleotide or protein sequences are differentially expressed, activated, inactivated or altered in cancers as compared to normal tissue. As outlined below, sequences include those that are up- regulated (i.e. expressed at a higher level), as well as those that are down- regulated (i.e. expressed at a lower level), in cancers. Sequences also include sequences that have been altered (i.e., truncated sequences or sequences with substitutions, deletions or insertions, including point mutations) and show either the same expression profile or an altered profile. In a preferred embodiment, the sequences are from humans; however, as will be appreciated by those in the art, sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, and farm animals (including sheep, goats, pigs, cows, horses, etc). In some cases, prokaryotic sequences may be useful. Sequences from other organisms may be obtained using the techniques outlined below. A "biological sample" encompasses any sample obtained from a living system or subject. The definition encompasses blood, serum, tissue, and other samples of biological origin that can be collected from a living system, subject or individual. In one embodiment, biological samples are obtained through sampling by minimally invasive or non-invasive approaches (e.g., urine collection, stool collection, blood drawing, needle aspiration, and other procedures involving minimal risk, discomfort or effort). Biological samples can be gaseous (e.g., exhaled breath). Biological samples are often liquid (sometimes referred to as a "biological fluid"). Liquid biological samples include, but are not limited to, urine, blood, interstitial fluid, edema fluid, saliva, lacrimal fluid, inflammatory exudates, synovial fluid, abscess, empyema or other infected fluid, cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminal fluid, feces, bile, intestinal secretions, and others. Biological samples include samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" also encompasses a clinical sample such as serum, plasma, other biological fluid, or tissue samples, and also includes cells in culture, cell supernatants and cell lysates.

[0024] As used herein, the term "biomarker" refers to a physical, biochemical, or physiologic measurement from or on the organism that represents a true or intended mechanistic target of a compound or a mechanistic event believed to be responsible for, or contributing in, a causal manner to the initiation, progression, severity, pathology, aggressiveness, grade, activity, disability, mortality, morbidity, disease sub-classification or other underlying pathogenic or pathologic feature of one or more diseases. A biomarker may be the target for monitoring the outcome of a therapeutic intervention (i.e., the functional or structural target of a drug agent). "Biomarker" refers to biochemical processes that are involved in, or are believed to be involved in, the etiology or progression of a disease or disorder. The biochemical process (i.e., the flow of molecules through a targeted metabolic pathway or network) is the focus of analysis (as disclosed herein) since it is the underlying changes of the biochemical process (i.e., molecular flux rates) that may be the significant or authentic target for treatment or diagnostic monitoring of the disease or disorder.

[0025] The terms "candidate agent" or "candidate compound" or "candidate molecule" or "candidate drug" as used herein is intended to encompass an agent, compound, or molecule which has the potential to have a therapeutic effect in vivo or in vitro which can be used with the disclosed methods to determine whether the agent or compound has a desired biological or biochemical activity.

[0026] The phrase "cellular characteristic associated with a proliferative disorder" as used herein is intended to include any feature or property, whether biological or biochemical, of a cell or cellular population that is indicative of a proliferative disorder, particularly that of NFl or an NFl related disease. For example, the characteristic may be but is not limited to, migration, proliferation, rate of cell growth, or cellular adhesion. The cellular characteristic may be that of individual cells or a population of cells.

[0027] As used herein, "chemical library" or "compound library" generally refers to a collection of stored chemicals often used in high-throughput screening or industrial manufacture. The library may comprise a series of stored chemicals, each chemical typically having associated information stored in a database. The associated information may include, for example, the chemical structure, purity, quantity, and physiochemical characteristics of the compound. Chemical or compound libraries may focus on large groups of varied organic chemical series such that an organic chemist can make many variations on the same molecular scaffold or molecular backbone. Chemicals may also be purchased from outside vendors as well and included into an internal chemical library.

[0028] The term "compound" as used herein (e.g., as in "test compound") is meant to include both exogenously added test compounds and peptides endogenously expressed from a peptide library. For example, in certain embodiments, the reagent cell also produces the test compound which is being screened. For instance, the reagent cell can produce, e.g., a test polypeptide, a test nucleic acid and/or a test carbohydrate which is screened for its ability to modulate the receptor/channel activity. In such embodiments, a culture of such reagent cells will collectively provide a library of potential effector molecules and those members of the library which either agonize or antagonize the receptor or ion channel function can be selected and identified. Moreover, it will be apparent that the reagent cell can be used to detect agents which transduce a signal via the receptor or channel of interest.

[0029] In other embodiments, the test compound is exogenously added. In such embodiments the test compound is contacted with the reagent cell. Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries. In such embodiments, both compounds which agonize or antagonize the receptor- or channel- mediated signaling function can be selected and identified.

[0030] By "derived from" is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, or combination thereof).

[0031] The terms "drug," "pharmaceutically active agent," "bioactive agent,"

"therapeutic agent," and "active agent" may be used interchangeably and refer to a substance, such as a chemical compound or complex, that has a measurable beneficial physiological effect on the body, such as a therapeutic effect in treatment of a disease or disorder, when administered in an effective amount. Further, when these terms are used, or when a particular active agent is specifically identified by name or category, it is understood that such recitation is intended to include the active agent per se, as well as pharmaceutically acceptable, pharmacologically active derivatives thereof, or compounds significantly related thereto, including without limitation, salts, pharmaceutically acceptable salts, N-oxides, prodrugs, active metabolites, isomers, fragments, analogs, solvates hydrates, radioisotopes, etc.

[0032] The phrase "effective amount" refers to that amount of a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The effective amount of such substance will vary depending upon the individual and disease condition being treated, the weight and age of the individual, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0033] An "individual" or "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0034] By "isolated DNA" is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0035] By a "mutation" is meant an alteration in sequence, either by site-directed or random mutagenesis. A mutated form of a protein encompasses point mutations as well as insertions, deletions, or rearrangements. A mutant is an organism containing a mutation. [0036] As used herein, the phrase "NFl -related disorder or condition" is intended to include any disease state or disorder or symptoms that result from or is associated with a mutation, deletion, dysregulation or other alteration of the NFl gene. Such disorders include, but are not limited to Neurofibromatosis Type I. The phrase "NFl -related disorder" may also encompass diseases wherein the etiology of which involves deregulation of RAS signaling. Associated conditions include but are not limited to neurofibromas, malignant peripheral nerve sheath tumors, optic gliomas, schwannomas, gliomas, leukemias, pheochromocytomas and non-tumor manifestations, including learning disorders, and other sarcomas.

[0037] The term "non-peptidic compound" is intended to encompass compounds that are comprised, at least in part, of molecular structures different from naturally-occurring L-amino acid residues linked by natural peptide bonds. However, "non-peptidic compounds" are intended to include compounds composed, in whole or in part, of peptidomimetic structures, such as D- amino acids, non-naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L- amino acid residues linked by natural peptide bonds. "Non-peptidic compounds" also are intended to include natural products.

[0038] As used herein, the phrase "pharmaceutically acceptable salt(s)" includes but is not limited to salts of acidic or basic groups that may be present in compounds identified using the methods of the present invention. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., l,l '-methylene- bis-(2-hydroxy-3-naphthoate)) salts. Compounds that include an amino moiety may form pharmaceutically or cosmetically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds that are acidic in nature are capable of forming base salts with various pharmacologically or cosmetically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts. The term "potential therapeutic agent" is intended to encompass any candidate agent that is determined, using the disclosed methods, to have an in vitro effect on test cells, as described herein. Such agent is identified via the methods described herein as having a potential beneficial or therapeutic effect on NFl or NFl -related disorders. The effect measured may vary, but generally comprises inhibition of viability, growth, proliferation, or migration of test cells; variations of the effect that can be measured will be recognized by one of ordinary skill in the art. Potential therapeutic agents, as used herein, are identified as having a desired effect in vitro, and are considered "hits" which may be subjected to further in vitro or in vivo evaluation to determine or optimize the therapeutic benefit, or, alternatively, may be used to identify derivative or analogous agents which may in turn be evaluated for an in vivo or in vitro therapeutic effect. [0040] As used herein, the terms "prevent," "preventing" and "prevention" refer to the prevention of the development, recurrence or onset of a disorder or one or more symptoms thereof resulting from the administration of one or more compounds identified in accordance the methods of the invention or the administration of a combination of such a compound and a known therapy for such a disorder.

[0041] The term "prophylactic" or "therapeutic" treatment is art-recognized and refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).

[0042] As used herein, the term "small molecule" and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heterorganic and/or organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. [0043] The phrase "therapeutic effect" is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and/or conditions in an animal or human. The phrase "therapeutically-effective amount" means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending upon the individual and disease condition being treated, the weight and age of the individual, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0044] As used herein, the terms "therapy" and "therapies" refer to any method, protocol and/or agent that can be used in the prevention, treatment, management or amelioration of a disease or disorder or one or more symptoms thereof. Similarly, as used herein, the terms "treat," "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and/or duration of a disorder or one or more symptoms thereof.

[0045] Detailed Description

[0046] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology and biochemistry, which are within the skill of the art. [0047] Many biological functions are accomplished by altering the expression of various genes through transcriptional (e.g., through control of initiation, provision of RNA precursors, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle, cell differentiation and cell death, are often characterized by the variations in the expression levels of groups of genes.

[0048] Changes in gene expression also are associated with pathogenesis. For example, the lack of sufficient expression of functional tumor suppressor genes and/or the over expression of oncogene/protooncogenes could lead to tumorigenesis or hyperplastic growth of cells. Thus, changes in the expression levels of particular genes (e.g., oncogenes or tumor suppressors) serve as signposts for the presence and progression of various diseases.

[0049] Monitoring changes in gene expression may also provide certain advantages during drug screening development. Often drugs are screened and prescreened for the ability to interact with a major target without regard to other effects the drugs have on cells. Often such other effects cause toxicity in the whole animal, which prevent the development and use of the potential drug.

[0050] The present inventors have examined tissue samples from neurofibromatosis and/or MPNST to identify the global changes in gene expression associated with neurofibromatosis and/or MPNST. These global changes in gene expression, also referred to as expression profiles, provide useful markers for diagnostic uses as well as markers that can be used to monitor disease states, disease progression, drug toxicity, drug efficacy and drug metabolism. [0051] The present invention provides compositions and methods to detect the level of expression of genes that may be differentially expressed dependent upon the state of the cell, i.e., normal versus cancerous and benign versus malignant. As used herein, the phrase "detecting the level expression" includes methods that quantitate expression levels as well as methods that determine whether a gene of interest is expressed at all. Thus, an assay which provides a yes or no result without necessarily providing quantification of an amount of expression is an assay that requires "detecting the level of expression" as that phrase is used herein.

[0052] Current care and clinical trials for patients with NFl -related disorder is hampered by a lack of biomarkers to predict and monitor response to therapy or progression of disease. Without biomarkers that allow prediction of a patient's responsiveness to current therapies, patients are offered therapies via an empiric approach, while subjecting many patients to unnecessary side effects and expense, further delaying recovery and improved quality of life. As such, identification of biomarkers offers the opportunity to define distinct immunogenetic sub-types of disease which may benefit from specific treatment approaches. Embedded within the gene signatures of neurofibromas and MPNSTs are biomarkers and molecular targets for therapeutic development in NFl, including SOX9.

[0053] The availability of sensitive biomarker permits the treating physician to evaluate a patient's likely responsiveness to first line or second line therapeutics. The prediction of the patient's likely response permits the physician to select therapies likely to be most efficacious for a given individual, avoiding treatment with less useful therapies. This, in turn, avoids subjecting the patient to unnecessary side effects and expense, while improving the patient's quality of life and creating opportunities to delay or prevent disease progression. [0054] The instant disclosure relates generally to compositions and methods of using such compositions for the identification of potential therapeutic agents useful for the treatment of NFl or NFl -related diseases. The disclosure also relates to the finding that a mammalian cell based system, wherein the cells are genetically modified to provide a novel and effective means for identifying potential therapeutic agents for the treatment of NFl or NFl -related disorders.

[0055] The invention is based on the discovery of a pattern of gene expression correlated with neurofibromatosis type 1 (NFl)-derived tumors. The genes that are differentially expressed in NFl are collectively referred to herein as "NFl nucleic acids" or "NFl polynucleotides" and the corresponding encoded polypeptides are referred to as "NFl polypeptides" or "NFl proteins."

[0056] The invention is also based on the discovery of a pattern of gene expression correlated with MPNST. The genes that are differentially expressed in MPNST are collectively referred to herein as "MPNST nucleic acids" or "MPNST polynucleotides" and the corresponding encoded polypeptides are referred to as "MPNST polypeptides" or "MPNST proteins."

[0057] Accordingly, the invention features a method of diagnosing a neurofibromatosis type 1 (NFl)-derived tumor or a predisposition to developing a neurofibromatosis type 1 (NFl)-derived tumor in a subject by determining an expression level of a NFl -associated gene in a patient derived biological sample, such as tissue sample. By NFl -associated gene is meant a gene that is characterized by an expression level which differs in a cell obtained from a subject having neurofibroma, malignant peripheral nerve sheath tumors (MPNST) and/or other peripheral nerve tumor or sarcoma compared to a normal cell. A normal cell is one obtained from testis tissue. A NFl -associated gene is one or more of the genes listed in Tables 1-3. An alteration, e.g., increase or decrease of the level of expression of the gene compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing neurofibroma, malignant peripheral nerve sheath tumors (MPNST) and/or other peripheral nerve tumor or sarcoma or primary benign neurofibromas.

[0058] By normal control level is meant a level of gene expression detected in a normal, healthy individual or in a population of individuals known not to be suffering from neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma. A control level is a single expression pattern derived from a single reference population or from a plurality of expression patterns. For example, the control level can be a database of expression patterns from previously tested cells. A normal individual is one with no clinical symptoms of neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma and without any family history of neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma.

[0059] An increase in the level of the genes listed in Tables 1-3 detected in a test sample compared to a normal control level indicates the subject (from which the sample was obtained) suffers from or is at risk of developing

NFl.

[0060] An increase in the level of the genes listed in Tables 1 detected in a test sample compared to a normal control level indicates the subject (from which the sample was obtained) suffers from or is at risk of developing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma.

[0061] An increase in the level of the genes listed in Table 2 detected in a test sample compared to a normal control level coupled with no increase in the level of the genes listed in Table 2 detected in a test sample compared to a normal control level indicates the subject (from which the sample was obtained) suffers from or is at risk of developing primary benign neurofibromas.

[0062] Alternatively, expression of a panel of NFl -associated genes in the sample is compared to a neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma control level of the same panel of genes. By MPNST control level is meant the expression profile of the NFl -associated genes found in a population suffering from MPNST.

[0063] Gene expression is increased or decreased 10%, 25%, 50% compared to the control level. Alternately, gene expression is increased or decreased 0.1, 0.2, 1, 2, 5, 10 or more fold compared to the control level. Expression is determined by detecting hybridization, e.g., on an array, of a NFl- associated gene probe to a gene transcript of the patient-derived tissue sample.

[0064] The patient derived tissue sample is any tissue from a test subject, e.g., a patient known to or suspected of having neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma. For example, the tissue contains a tumor cell. For example, the tissue is a cell from nerve cells.

[0065] The invention also provides a neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma reference expression profile of a gene expression level of two or more of the genes listed in Tables 1-3. Alternatively, the invention provides a MPNST reference expression profile of the levels of expression of two or more of the genes listed in Tables 1-3 or the genes listed in Tables 1-3.

[0066] The invention further provides methods of identifying an agent that inhibits or enhances the expression or activity of a NFl -associated gene, e.g. the genes listed in Tables 1-3 by contacting a test cell expressing a neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma associated gene with a test agent and determining the expression level of the neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma associated gene. A decrease of the level compared to a normal control level of the gene indicates that the test agent is an inhibitor of the NFl- associated gene and reduces a symptom of neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma. Alternatively, an increase of the level or activity compared to a normal control level or activity of the gene indicates that the test agent is an enhancer of expression or function of the neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma associated gene.

[0067] The invention also provides a kit with a detection reagent which binds to two or more MPNST nucleic acid sequences or which binds to a gene product encoded by the nucleic acid sequences. Also provided is an array of nucleic acids that binds to two or more MPNST nucleic acids.

[0068] Therapeutic methods include a method of treating or preventing MPNST in a subject by administering to the subject an antisense composition. The antisense composition reduces the expression of a specific target gene, e.g., the antisense composition contains a nucleotide, which is complementary to a sequence selected from the group consisting of the genes listed in Tables 1-3. Another method includes the steps of administering to a subject a short interfering RNA (siRNA) composition. The siRNA composition reduces the expression of a nucleic acid selected from the group consisting of the genes listed in Tables 1-3.

[0069] In yet another method, treatment or prevention of MPNST in a subject is carried out by administering to a subject a ribozyme composition. The nucleic acid-specific ribozyme composition reduces the expression of a nucleic acid selected from the group consisting of the genes listed in Tables 1-3. Other therapeutic methods include those in which a subject is administered a compound that increases the expression of the genes listed in Tables 1-3 or activity of a polypeptide encoded by the genes listed in Tables 1-3. Furthermore, MPNST can be treated by administering a protein encoded by the genes listed in Tables 1-3. The protein may be directly administered to the patient or, alternatively, may be expressed in vivo subsequent to being introduced into the patient, for example, by administering an expression vector or host cell carrying the down- regulated marker gene of interest. Suitable mechanisms for in vivo expression of a gene of interest are known in the art.

[0070] In one embodiment, SOX9, an HMG-box containing transcription factor, is a key marker of neural crest and is expressed at only very low levels in normal Schwann cells. The present invention demonstrates a role for SOX9 in growth rate control in neurofibroma and as a factor to which MPNST are strictly addicted, undergoing cell death upon diminution. Till now, SOX9 protein has only been known as a cause of some forms of dwarfism and acts as a tumor suppressor in some prostate and colon cancers. In stark contrast to the previous studies, in NFl we find a large increase in SOX9 mRNA. The present invention validates the array data in human tissue sections: SOX9 protein was detected in 42/42 neurofibroma and MPNST tissue sections. And critically, reducing SOX9 expression slowed growth of neurofibroma cells and killed MPNST cells.

[0071] Comparing gene expression in primary cells is useful for identifying genes in tumors that are potential biomarkers and/or therapeutic targets. The present invention identifies SOX9, a novel biomarker of NFl -related tumors, which is necessary for malignant tumor cell survival. The present invention reveals progressive failure of Schwann cell differentiation from benign to malignant NFl tumor samples, similarity of dermal and plexiform neurofibromas, and candidate regions for potential chromosomal amplifications and deletions shared across cell culture and tumor comparisons.

[0072] The invention includes methods of diagnosing the presence or absence of neurofibromatosis and/or MPNST in a patient comprising the step of detecting the level of expression in a tissue sample of two or more genes from Tables 1-3; wherein differential expression of the genes in Tables 1- 3 is indicative of neurofibromatosis and/or MPNST. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1.

[0073] The invention also includes methods of detecting the progression of neurofibromatosis and/or MPNST and/or differentiating benign from malignant disease. For instance, methods of the invention include detecting the progression of neurofibromatosis and/or MPNST in a patient comprising the step of detecting the level of expression in a tissue sample of two or more genes from Tables 1-3; wherein differential expression of the genes in Tables 1-3 is indicative of neurofibromatosis and/or MPNST progression. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1.

[0074] In some aspects, the present invention provides a method of monitoring the treatment of a patient with neurofibromatosis and/or MPNST, comprising administering a pharmaceutical composition to the patient and preparing a gene expression profile from a cell or tissue sample from the patient and comparing the patient gene expression profile to a gene expression from a cell population comprising normal cells or to a gene expression profile from a cell population comprising neurofibromatosis and/or MPNST cells or to both. In some preferred embodiments, the gene profile will include the expression level of one or more genes in Tables 1- 3. In other preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1.

[0075] In another aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene in Tables 1-3, preparing a gene expression profile from a cell or tissue sample from the patient comprising tumor cells and comparing the patient expression profile to a gene expression profile from an untreated cell population comprising neurofibromatosis and/or MPNST cells. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1.

[0076] In one aspect, the present invention provides a method of diagnosing neurofibromatosis and/or MPNST in a patient, comprising detecting the level of expression in a tissue sample of two or more genes from Tables 1- 3, wherein differential expression of the genes in Tables 1-3 is indicative of neurofibromatosis and/or MPNST. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1-3.

[0077] In another aspect, the present invention provides a method of detecting the progression of neurofibromatosis and/or MPNST in a patient, comprising detecting the level of expression in a tissue sample of two or more genes from Tables 1-3; wherein differential expression of the genes in Tables 1- 3 is indicative of neurofibromatosis and/or MPNST progression. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1.

[0078] The present invention also provides materials and methods for monitoring the treatment of a patient with a neurofibromatosis and/or MPNST. The present invention provides a method of monitoring the treatment of a patient with neurofibromatosis and/or MPNST, comprising administering a pharmaceutical composition to the patient, preparing a gene expression profile from a cell or tissue sample from the patient and comparing the patient gene expression profile to a gene expression from a cell population comprising normal cells or to a gene expression profile from a cell population comprising neurofibromatosis and/or MPNST cells or to both. In some preferred embodiments, the method may include detecting the level of expression of one or more genes from the genes listed in Tables 1- 3. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1-3.

[0079] In a related aspect, the present invention provides a method of treating a patient with neurofibromatosis and/or MPNST, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene in Tables 1-3, preparing a gene expression profile from a cell or tissue sample from the patient comprising neurofibromatosis and/or MPNST cells and comparing the patient expression profile to a gene expression profile from an untreated cell population comprising neurofibromatosis and/or MPNST cells. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1. [0080] The present invention provides a method of diagnosing a malignant tumor in a patient, comprising detecting the level of expression in a tissue sample of two or more genes from Tables 1-3, wherein differential expression of the genes in Tables 1-3 is indicative of neurofibromatosis and/or MPNST. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0081] The present invention provides a method of detecting the progression of a malignant tumor in a patient, comprising detecting the level of expression in a tissue sample of two or more genes from Tables 1-3, wherein differential expression of the genes in Tables 1-3 is indicative of a malignant tumor progression. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0082] In a related aspect, the present invention provides a method of monitoring the treatment of a patient with a malignant tumor, comprising administering a pharmaceutical composition to the patient, preparing a gene expression profile from a cell or tissue sample from the patient and comparing the patient gene expression profile to a gene expression from a cell population comprising benign tumor cells or to a gene expression profile from a cell population comprising malignant tumor cells or to both. In some preferred embodiments, the method of the present invention may include detecting the expression level of one or more genes selected from the genes listed in Tables 1-3. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0083] In some preferred embodiments, the present invention provides a method of treating a patient with a malignant tumor, comprising administering to the patient a pharmaceutical composition, wherein the composition alters the expression of at least one gene in Tables 1-3, preparing a gene expression profile from a cell or tissue sample from the patient comprising malignant tumor cells and comparing the patient expression profile to a gene expression profile from an untreated cell population comprising malignant tumor cells. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Tables 1-3.

[0084] The invention also includes methods of differentiating malignant neurofibromatosis and/or MPNST from neurofibromatosis and/or MPNST in a patient comprising the step of detecting the level of expression in a tissue sample of two or more genes from Tables 1-3; wherein differential expression of the genes in Tables 1-3 is indicative of malignant neurofibromatosis and/or MPNST rather than neurofibromatosis and/or MPNST.

[0085] The invention further includes methods of screening for an agent capable of modulating the onset or progression of neurofibromatosis and/or MPNST, comprising the steps of exposing a cell to the agent; and detecting the expression level of two or more genes from Tables 1-3. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0086] Any of the methods of the invention described above may include the detection of at least 2 genes from the tables. Preferred methods may detect all or nearly all of the genes in the tables. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0087] The invention further includes compositions comprising at least two oligonucleotides, wherein each of the oligonucleotides comprises a sequence that specifically hybridizes to a gene in Tables 1-3 as well as solid supports comprising at least two probes, wherein each of the probes comprises a sequence that specifically hybridizes to a gene in Tables 1-3. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0088] The invention further includes computer systems comprising a database containing information identifying the expression level in NFl related tissue of a set of genes comprising at least two genes in Tables 1-3; and a user interface to view the information. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1. The database may further include sequence information for the genes, information identifying the expression level for the set of genes in normal, benign and malignant tissue (malignant and nonmalignant) and may contain links to external databases such as GenBank.

[0089] The invention further comprises kits useful for the practice of one or more of the methods of the invention. In some preferred embodiments, a kit may contain one or more solid supports having attached thereto one or more oligonucleotides. The solid support may be a high-density oligonucleotide array. Kits may further comprise one or more reagents for use with the arrays, one or more signal detection and/or array-processing instruments, one or more gene expression databases and one or more analysis and database management software packages.

[0090] Lastly, the invention includes methods of using the databases, such as methods of using the disclosed computer systems to present information identifying the expression level in a tissue or cell of at least one gene in Tables 1-3, comprising the step of comparing the expression level of at least one gene in Tables 1-3 in the tissue or cell to the level of expression of the gene in the database. In some preferred embodiments, one or more genes may be selected from a group consisting of the genes listed in Table 1.

[0091] In another embodiment, the present invention provides for methods for detecting, or for detecting and distinguishing between or among cell proliferative disorders in a subject, comprising determining, in a biological sample isolated from a subject, the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund "^■ (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes, respectively.

[0092] In another embodiment, the present invention provides for methods for detecting, or for detecting and distinguishing between or among cell proliferative disorders in a subject, comprising determining, in a biological sample isolated from a subject, the expression levels of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIXl -4 genes, respectively.

[0093] In another embodiment, the present invention provides for a method for detecting, or for detecting and distinguishing between or among malignant peripheral nerve sheath tumor (MPNST) cell proliferative disorders in a subject, comprising determining, in a biological sample isolated from a subject, the expression levels of the EYA4 gene.

[0094] In another embodiment, an increase in expression level of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIXl- 4 genes is determinative of a malignant peripheral nerve sheath tumor (MPNST) cell proliferative disorder in a subject. In another embodiment, a three-fold increase in expression level of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIXl -4 genes is determinative of a malignant peripheral nerve sheath tumor (MPNST) cell proliferative disorder in a subject.

[0095] In one embodiment, the expression level is determined by detecting the presence, absence or level of mRNA transcribed from the gene or sequence. In another embodiment, the expression level is determined by detecting the presence, absence or level of a polypeptide encoded by the gene or sequence.

[0096] In another embodiment, the method further comprises comparing the expression level to a normal standard wherein a decrease in DACHl expression compared to normal expression indicates that the subject is a candidate for further examination for cancer. In another embodiment, the method further comprises comparing the expression level to a normal standard wherein an increase in expression of one or more of EYAl, EYA2, EYA4, and SIXl -4 compared to normal expression indicates that the subject is a candidate for further examination for cancer.

[0097] In another embodiment, the body fluid is selected from blood, plasma, serum, lymph, ascitic fluid, gynecological fluid, urine, a fluid collected by vaginal rinsing, a saliva specimen, and a fluid collected by mouth rinsing.

[0098] In another embodiment, the present invention provides for a method for identifying an agent as a candidate for treating cancer, the method comprising the steps of: exposing cancer cells to a test agent; measuring the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes; and comparing the expression level to that of control cells not exposed to the test agent wherein a lower or higher than control expression indicates that the agent is a candidate for treating cancer.

[0099] In another embodiment, the increase in Dachshund (DACH) expression compared to control expression indicates that the agent is a candidate for treating cancer. In another embodiment, the decrease in the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Sine Oculis (SIX) and paired box-containing (PAX) compared to control expression indicates that the agent is a candidate for treating cancer. In another embodiment, the increase in DACHl expression compared to control expression indicates that the agent is a candidate for treating cancer. In another embodiment, a decrease in the expression levels of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIX1-4 compared to control expression indicates that the agent is a candidate for treating cancer. In another embodiment, the decrease in EY A4 expression compared to control expression indicates that the agent is a candidate for treating cancer.

[00100] In another embodiment, the present invention provides for a method for determining the effectiveness of a treatment for cancer, the method comprising the steps of: measuring the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes in a first sample from a cancer patient prior to providing at least a portion of the treatment to the patient; measuring the expression of the expression levels in a second sample from the patient after the portion of the treatment is provided to the patient; and comparing the expression levels of the first sample and the second sample wherein a modified expression level in the second sample indicates that the treatment is effective. [00101] In another embodiment, the present invention provides for a method for determining the effectiveness of a treatment for cancer, the method comprising the steps of: measuring the expression levels of one or more genes selected from the group consisting of DACHl, EYAl, EYA2, EYA4, and SIXl -4 genes in a first sample from a cancer patient prior to providing at least a portion of the treatment to the patient; measuring the expression of the expression levels in a second sample from the patient after the portion of the treatment is provided to the patient; and comparing the expression levels of the first sample and the second sample wherein an increase in DACHl expression and/or a decrease in the expression of one or more of EYAl, EYA2, EYA4, and SIX1-4 expression level in the second sample indicates that the treatment is effective.

[00102] In another embodiment, the present invention provides for a method for treating or preventing cancer in a human or non-human animal comprising the step of: administering to the human or non-human animal having cancer an active agent in an amount effective for treating cancer wherein the active agent comprises a therapeutic agent effective to increase DACHl expression and/or decrease expression of one or more of EYAl, EYA2, EYA4, and SIX1-4.

[00103] In another embodiment, the present invention provides for a method for treating or preventing cancer in a human or non-human animal comprising the step of: administering to the human or non-human animal having cancer an active agent in an amount effective for treating cancer wherein the active agent comprises a combination of a therapeutic agent for cancer and a therapeutic agent effective to increase DACHl expression and/or decrease expression of one or more of EYAl, EYA2, EYA4, and SIXl -4. [00104] In another embodiment, the present invention provides for a diagnostic method for malignant peripheral nerve sheath tumors (MPNST) comprising: (a) determining in a sample from a patient an expression level of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes, (b) comparing the patient's expression level to the expression level in a normal subject, and (c) diagnosing the patient as at risk for MPNST when the patient's expression level is significantly increased or decreased compared to the normal subject's expression level.

[00105] In another embodiment, the present invention provides for a diagnostic method for malignant peripheral nerve sheath tumors (MPNST) comprising: (a) determining in a sample from a patient an expression level of one or more genes selected from the group consisting of DACHl, EYAl, EYA2, EYA4, and SIX1-4 genes, (b) comparing the patient's expression level to the expression level in a normal subject, and (c) diagnosing the patient as at risk for MPNST when the patient's DACHl expression is significantly decreased compared to the normal subject's expression level and/or the patient's expression of one or more of EYAl, EYA2, EYA4, and SIX1-4 is significantly increased compared to the normal subject's expression level. In one embodiment, the expression level in the patient is increased or decreased about 3 -fold.

[00106] In another embodiment, the present invention provides for a diagnostic method for malignant peripheral nerve sheath tumors (MPNST) comprising:(a) determining in a sample from a patient an expression level OΪEYA4, (b) comparing the patient's expression level to the EYA4 expression level in a normal subject, and (c) diagnosing the patient as at risk for MPNST when the patient's EYA4 expression is significantly increased compared to the normal subject's expression level. In one embodiment, the EYA4 expression level in the patient is about 2 to about 20-fold increased, as compared to that in the normal subject. In another embodiment, the EYA4 expression level in the patient is about 3 to about 70-fold increased, as compared to that in the normal subject. In another embodiment, the EYA4 expression level in the patient is about 5 to about 90-fold increased, as compared to that in the normal subject.

[00107] In another embodiment, the present invention provides for a method of a treatment for malignant peripheral nerve sheath tumors (MPNST) comprising administering to a patient in need thereof a biologically effective amount of one or more therapeutic agents capable of directly or indirectly modulating expression or activity levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes.

[00108] In another embodiment, the present invention provides for a method of a treatment for malignant peripheral nerve sheath tumors (MPNST) comprising administering to a patient in need thereof a biologically effective amount of one or more therapeutic agents capable of directly or indirectly increasing the expression or activity levels of DACHl and/or directly or indirectly decreasing the expression or activity levels of one or more of EYAl, EYA2, EYA4, and SIXl -4.

[00109] In another embodiment, the present invention provides for a method of a treatment for malignant peripheral nerve sheath tumors (MPNST) comprising administering to a patient in need thereof a biologically effective amount of one or more therapeutic agents capable of directly or indirectly decreasing the expression or activity levels of EYA4.

[00110] In one embodiment, the therapeutic agent comprises oligonucleotides, antisense oligonucleotides, polynucleotides, therapeutic DNA, ribozymes, dsRNAs, siRNA, RNAi, or gene therapy vectors. In another embodiment, the therapeutic agents comprise EYA4 inhibitors, antagonists, antibodies or other immunologically active molecules that immunoreact with EYA4 protein, resulting in apoptosis of MPNST cells. In another embodiment, the therapeutic agents comprise EK44inhibitors, antagonists, antibodies or other immunologically active molecules that immunoreact with EYA4 protein in combination with one or more additional cancer therapeutic agent, resulting in apoptosis of MPNST cells.

[00111] In one embodiment, the expression of a marker can be assessed at the protein level using an antibody (e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled or enzyme-labeled antibody) or an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)) that binds specifically to the marker protein or fragment thereof. For example, en2yme linked immunosorbent assays (ELISAs), Western blot analysis and in situ hybridizations can be employed for this purpose.

[00112] In another embodiment, the expression of a marker can be assessed at the mRNA level by preparing and detecting/measuring mRNA/cDNA from cells. For example, RT-PCR (e.g., quantitative RT-PCR), Southern blot analysis, Northern blot analysis, and in situ hybridizations can be used for this purpose. It is well within the capability of one of ordinary skill in the art to design primers and probes for assessing the expression of a marker at the mRNA level.

[00113] The differentially expressed genes identified herein are used for diagnostic purposes as markers of MPNST and as gene targets, the expression of which is altered to treat or alleviate a symptom of MPNST. By measuring expression of the various genes in a sample of cells, MPNST is diagnosed. Similarly, by measuring the expression of these genes in response to various agents, and agents for treating MPNST can be identified.

[00114] Using sequence information provided by the GENEBANK database entries for the known sequences the MPNST associated genes are detected and measured using techniques well known to one of ordinary skill in the art. For example, sequences within the sequence database entries corresponding to MPNST sequences, are used to construct probes for detecting MPNST RNA sequences in, e.g., northern blot hybridization analyses. Probes include at least 10, 20, 50, 100, 200 nucleotides of a reference sequence. As another example, the sequences can be used to construct primers for specifically amplifying the MPNST sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction.

[00115] Expression level of one or more of the MPNST sequences in the test cell population, e.g., a patient derived tissues sample is then compared to expression levels of the some sequences in a reference population. The reference cell population includes one or more cells for which the compared parameter is known, i.e., MPNST cells or non-MPNST cells.

[00116] Whether or not a pattern of gene expression in the test cell population compared to the reference cell population indicates neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma or a predisposition thereto depends upon the composition of the reference cell population. For example, if the reference cell population is composed of non-MPNST cells, a similar gene expression pattern in the test cell population and reference cell population indicates the test cell population is non-MPNST. Conversely, if the reference cell population is made up of MPNST cells, a similar gene expression profile between the test cell population and the reference cell population indicates that the test cell population includes MPNST cells.

[00117] A level of expression of a MPNST marker gene in a test cell population is considered altered in levels of expression if its expression level varies from the reference cell population by more than 1.0, 1.5, 2.0, 5.0, 10.0 or more fold from the expression level of the corresponding MPNST sequence in the reference cell population.

[00118] Differential gene expression between a test cell population and a reference cell population is normalized to a control nucleic acid, e.g. a housekeeping gene. For example, a control nucleic acid is one which is known not to differ depending on the endometriotic or non-endometriotic state of the cell. Expression levels of the control nucleic acid in the test and reference nucleic acid can be used to normalize signal levels in the compared populations. Control genes include beta-actin, glyceraldehyde 3-phosphate dehydrogenase or ribosomal protein Pl.

[00119] The test cell population is compared to multiple reference cell populations.

Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a second reference cell population known to contain, e.g., MPNST cells, as well as a second reference population known-to contain, e.g., non-MPNST cells (normal cells). The test cell is included in a tissue type or cell sample from a subject known to contain, or to be suspected of containing, MPNST cells.

[00120] The test cell is obtained from a bodily tissue or a bodily fluid, e.g., biological fluid (such as blood or urine). For example, the test cell is purified from a tissue. In one embodiment, the test cell population comprises a Schwann cell (also referred to as neurolemmocytes). The Schwann cell is from tissue known to be or suspected to be a MPNST.

[00121] Cells in the reference cell population are derived from a tissue type as similar to test cell. Optionally, the reference cell population is a cell line, e.g., a MPNST cell line (positive control) or a normal non-MPNST cell line (negative control). Alternatively, the control cell population is derived from a database of molecular information derived from cells for which the assayed parameter or condition is known.

[00122] Expression of the genes disclosed herein is determined at the protein or nucleic acid level using methods known in the art. For example, Northern hybridization analysis using probes which specifically recognize one or more of these sequences can be used to determine gene expression. Alternatively, expression is measured using reverse-transcription-based PCR assays, e.g., using primers specific for the differentially expressed sequences. Expression is also determined at the protein level, i.e., by measuring the levels of polypeptides encoded by the gene products described herein, or biological activity thereof. Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes. The biological activity of the proteins encoded by the genes is also well known.

[00123] Diagnosing MPNST

[00124] MPNST is diagnosed by measuring the level of expression of one or more

MPNST nucleic acid sequences from a test population of cells, (i.e., a patient derived biological sample). Gene expression is also measured from blood or other bodily fluids such as urine. Other biological samples can be used for measuring the protein level. For example, the protein level in the blood, or serum derived from subject to be diagnosed can be measured by immunoassay or biological assay.

[00125] Expression of one or more of NFl -associated genes, e.g., the genes listed in Tables 1-3 is determined in the test cell or biological sample and compared to the expression of the normal control level. A normal control level is an expression profile of NFl -associated genes typically found in a population known not to be suffering from neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma. An increase or a decrease of the level of expression in the patient derived tissue sample of the MPNST associated genes indicates that the subject is suffering from or is at risk of developing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma. For example, an increase in expression of the genes listed in Tables 1-3 in the test population compared to the normal control level indicates that the subject is suffering from or is at risk of developing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma.

[00126] When one or more of the neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma -associated genes are altered in the test population compared to the normal control level indicates that the subject suffers from or is at risk of developing MPNST. For example, at least 1%, 5%, 25%, 50%, 60%, 80%, 90% or more of the panel of NFl -associated genes (the genes listed in Tables 1-3, the genes listed in Tables 1-3, or the genes listed in Tables 1-3) are altered.

[00127] Identifying Agents that Inhibit NF 1 -associated Gene Expression

[00128] An agent that inhibits the expression or activity of a NF 1 -associated gene is identified by contacting a test cell population expressing a NFl associated up-regulated gene with a test agent and determining the expression level of the NFl associated gene. A decrease in expression in the presence of the agent compared to the normal control level (or compared to the level in the absence of the test agent) indicates the agent is an inhibitor of a NFl associated up-regulated gene and useful to inhibit NFl.

[00129] Alternatively, an agent that decreases the expression or activity of a

MPNST up-regulated associated gene is identified by contacting a test cell population expressing a MPNST associated gene with a test agent and determining the expression level or activity of the MPNST associated down-regulated gene. A decrease of expression or activity compared to a normal control expression level or activity of the NFl -associated gene indicates that the test agent is an inhibitor of expression or activity of the up-regulated MPNST associated gene.

[00130] The test cell population is any cell expressing the NFl -associated genes.

For example, the test cell population contains an epithelial cell. For example, the test cell is an immortalized cell line derived from a tumor. Alternatively, the test cell is a cell, which has been transfected with a NFl -associated gene or which has been transfected with a regulatory sequence (e.g. promoter sequence) from a NFl -associated gene operably linked to a reporter gene.

[00131] Assessing Efficacy of Treatment of MPNST in a Subject

[00132] The differentially expressed MPNST sequences identified herein also allow for the course of treatment of MPNST to be monitored. In this method, a test cell population is provided from a subject undergoing treatment for MPNST. If desired, test cell populations are obtained from the subject at various time points before, during, or after treatment. Expression of one or more of the MPNST sequences, in the cell population is then determined and compared to a reference cell population which includes cells whose MPNST state is known. The reference cells have not been exposed to the treatment.

[00133] If the reference cell population contains no MPNST cells, a similarity in expression between MPNST sequences in the test cell population and the reference cell population indicates that the treatment is efficacious. However, a difference in expression between MPNST sequences in the test population and a normal control reference cell population indicates the less favorable clinical outcome or prognosis.

[00134] By "efficacious" is meant that the treatment leads to a reduction in expression of a pathologically up-regulated gene, increase in expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of in a subject. When treatment is applied prophylactically, "efficacious" means that the treatment retards or prevents MPNST from forming or retards, prevents, or alleviates a symptom of clinical MPNST. Assessments are made using standard clinical protocols.

[00135] Efficaciousness is determined in association with any known method for diagnosing or treating MPNST. MPNST is diagnosed for example, by identifying symptomatic anomalies, e.g., pain within neurofibroma.

[00136] Selecting a Therapeutic Agent for Treating MPNST that is Appropriate for a Particular Individual

[00137] Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. An agent that is metabolized in a subject to act as an anti-MPNST agent can manifest itself by inducing a change in gene expression pattern in the subject's cells from that characteristic of an MPNST state to a gene expression pattern characteristic of a non-MPNST state. Accordingly, the differentially expressed MPNST sequences disclosed herein allow for a putative therapeutic or prophylactic inhibitor of MPNST to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable inhibitor of MPNST in the subject.

[00138] To identify an inhibitor or enhancer of MPNST, that is appropriate for a specific subject, a test cell population from the subject is exposed to a therapeutic agent, and the expression of one or more of the genes listed in Tables 1 -3 sequences is determined.

[00139] The test cell population contains a MPNST cell expressing a MPNST associated gene. Preferably, the test cell is a Schwann cell. For example a test cell population is incubated in the presence of a candidate agent and the pattern of gene expression of the test sample is measured and compared to one or more reference profiles, e.g., a MPNST reference expression profile or a non-MPNST reference expression profile.

[00140] A decrease in expression of one or more of the sequences the genes listed in Tables 1-3 or an increase in expression of one or more of the sequences the genes listed in Tables 1-3 in a test cell population relative to a reference cell population containing MPNST is indicative that the agent is therapeutic.

[00141] The test agent can be any compound or composition. For example, the test agents are immunomodulatory agents.

[00142] Assay Formats

[00143] The genes identified as being differentially expressed in neurofibromatosis and/or MPNST may be used in a variety of nucleic acid detection assays to detect or quantitate the expression level of a gene or multiple genes in a given sample. For example, traditional Northern blotting, nuclease protection, RT-PCR and differential display methods may be used for detecting gene expression levels. Those methods are useful for some embodiments of the invention. However, methods and assays of the invention are most efficiently designed with array or chip hybridization- based methods for detecting the expression of a large number of genes.

[00144] Any hybridization assay format may be used, including solution-based and solid support-based assay formats. Solid supports containing oligonucleotide probes for differentially expressed genes of the invention can be filters, polyvinyl chloride dishes, silicon or glass based chips, etc. Such wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755). Any solid surface, to which oligonucleotides can be bound, either directly or indirectly, either covalently or non-covalently, can be used. A preferred solid support is a high density array or DNA chip. These contain a particular oligonucleotide probe in a predetermined location on the array. Each predetermined location may contain more than one molecule of the probe, but each molecule within the predetermined location has an identical sequence. Such predetermined locations are termed features. There may be, for example, about 2, 10, 100, 1000 to 10,000; 100,000 or 400,000 of such features on a single solid support. The solid support or the area within which the probes are attached may be on the order of a square centimeter.

[00145] Oligonucleotide probe arrays for expression monitoring can be made and used according to any techniques known in the art (see for example, Lockhart et al., (1996) Nat. Biotechnol. 14, 1675-1680; McGaIl et al., (1996) Proc. Nat. Acad. Sci. USA 93, 13555-13460). Such probe arrays may contain at least two or more oligonucleotides that are complementary to or hybridize to two or more of the genes described herein. Such arrays may also contain oligonucleotides that are complementary or hybridize to at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70, 100 or more the genes described herein.

[00146] The genes which are assayed according to the present invention are typically in the form of mRNA or reverse transcribed mRNA. The genes may be cloned or not and the genes may be amplified or not. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+RNA as a source, as it can be used with less processing steps.

[00147] The sequences of the expression marker genes are in the public databases.

Tables 1-3 provide the GenBank accession number for the genes identified called either Accession # or Fragment Name. The sequences of the genes in GenBank are expressly incorporated by reference as are equivalent and related sequences present in GenBank or other public databases. The term "SEQ ID" refers to the sequence identification number correlating the listed gene to its sequence information as provided within the sequence listing of this application.

[00148] Probes based on the sequences of the genes described herein may be prepared by any commonly available method. Oligonucleotide probes for assaying the tissue or cell sample are preferably of sufficient length to specifically hybridize only to appropriate, complementary genes or transcripts. Typically the oligonucleotide probes will be at least 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some cases longer probes of at least 30, 40, or 50 nucleotides will be desirable.

[00149] As used herein, oligonucleotide sequences that are complementary to one or more of the genes described herein refers to oligonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequence of said genes. Such hybridizable oligonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes.

[00150] The phrase "hybridizing specifically to" refers to the binding, duplexing or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[00151] Assays and methods of the invention may utilize available formats to simultaneously screen at least about 100, preferably about 1000, more preferably about 10,000 and most preferably about 1,000,000 or more different nucleic acid hybridizations.

[00152] The term "mismatch control" or "mismatch probe" refer to a probe whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence. For each mismatch (MM) control in a high- density array, a corresponding perfect match (PM) probe that is perfectly complementary to the same particular target sequence typically exists. The mismatch may comprise one or more bases.

[00153] While the mismatch(s) may be located anywhere in the mismatch probe, terminal mismatches are less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequence. In a particularly preferred embodiment, the mismatch is located at or near the center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions.

[00154] The term "perfect match probe" refers to a probe that has a sequence that is perfectly complementary to a particular target sequence. The test probe is typically perfectly complementary to a portion (subsequence) of the target sequence. The perfect match (PM) probe can be a "test probe", a "normalization control" probe, an expression level control probe and the like. A perfect match control or perfect match probe is, however, distinguished from a "mismatch control" or "mismatch probe."

[00155] As used herein a "probe" is defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, U, C or T) or modified bases (7- deazaguanosine, inosine, etc.). In addition, the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.

[00156] The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but with only insubstantial hybridization to other sequences or to other sequences such that the difference may be identified. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5⁰C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.

[00157] Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O⁰C. for short probes (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[00158] The "percentage of sequence identity" or "sequence identity" is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical monomer unit (e.g., nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Percentage sequence identity when calculated using the programs GAP or BESTFIT (see below) is calculated using default gap weights.

[00159] Homology or identity may be determined by BLAST (Basic Local

Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268 and Altschul, (1993) J. MoI. Evol. 36, 290-300, fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a pre-selected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al., (1994) Nature Genet. 6, 119-129, which is filly incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters were adjusted as follows: Q=IO (gap creation penalty); R=IO (gap extension penalty); wink=l (generates word hits at every WINK position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=l; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

[00160] Probe Design

[00161] One of skill in the art will appreciate that an enormous number of array designs are suitable for the practice of this invention. The high density array will typically include a number of probes that specifically hybridize to the sequences of interest. See WO 99/32660 for methods of producing probes for a given gene or genes. In addition, in a preferred embodiment, the array will include one or more control probes.

[00162] High density array chips of the invention include "test probes." Test probes may be oligonucleotides that range from about 5 to about 500 or about 5 to about 50 nucleotides, more preferably from about 10 to about 40 nucleotides and most preferably from about 15 to about 40 nucleotides in length. In other particularly preferred embodiments the probes are about 20 to 25 nucleotides in length. In another preferred embodiment, test probes are double or single strand DNA sequences. DNA sequences are isolated or cloned from natural sources or amplified from natural sources using natural nucleic acid as templates. These probes have sequences complementary to particular subsequences of the genes whose expression they are designed to detect. Thus, the test probes are capable of specifically hybridizing to the target nucleic acid they are to detect.

[00163] In addition to test probes that bind the target nucleic acid(s) of interest, the high density array can contain a number of control probes. The control probes fall into three categories referred to herein as (1) normalization controls; (2) expression level controls; and (3) mismatch controls.

[00164] Normalization controls are oligonucleotide or other nucleic acid probes that are complementary to labeled reference oligonucleotides or other nucleic acid sequences that are added to the nucleic acid sample. The signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the signal of a perfect hybridization to vary between arrays. In a preferred embodiment, signals (e.g., fluorescence intensity) read from all other probes in the array are divided by the signal (e.g., fluorescence intensity) from the control probes thereby normalizing the measurements.

[00165] Virtually any probe may serve as a normalization control. However, it is recognized that hybridization efficiency varies with base composition and probe length. Preferred normalization probes are selected to reflect the average length of the other probes present in the array; however, they can be selected to cover a range of lengths. The normalization control(s) can also be selected to reflect the (average) base composition of the other probes in the array, however in a preferred embodiment, only one or a few probes are used and they are selected such that they hybridize well (i.e., no secondary structure) and do not match any target-specific probes.

[00166] Expression level controls are probes that hybridize specifically with constitutively expressed genes in the biological sample. Virtually any constitutively expressed gene provides a suitable target for expression level controls. Typical expression level control probes have sequences complementary to subsequences of constitutively expressed "housekeeping genes" including, but not limited to the β-actin gene, the transferrin receptor gene, the GAPDH gene, and the like.

[00167] Mismatch controls may also be provided for the probes to the target genes, for expression level controls or for normalization controls. Mismatch controls are oligonucleotide probes or other nucleic acid probes identical to their corresponding test or control probes except for the presence of one or more mismatched bases. A mismatched base is a base selected so that it is not complementary to the corresponding base in the target sequence to which the probe would otherwise specifically hybridize. One or more mismatches are selected such that under appropriate hybridization conditions (e.g., stringent conditions) the test or control probe would be expected to hybridize with its target sequence, but the mismatch probe would not hybridize (or would hybridize to a significantly lesser extent). Preferred mismatch probes contain a central mismatch. Thus, for example, where a probe is a twenty-mer, a corresponding mismatch probe will have the identical sequence except for a single base mismatch (e.g., substituting a G, a C or a T for an A) at any of positions 6 through 14 (the central mismatch). [00168] Mismatch probes thus provide a control for non-specific binding or cross hybridization to a nucleic acid in the sample other than the target to which the probe is directed. Mismatch probes also indicate whether hybridization is specific or not. For example, if the target is present the perfect match probes should be consistently brighter than the mismatch probes. In addition, if all central mismatches are present, the mismatch probes can be used to detect a mutation. The difference in intensity between the perfect match and the mismatch probe (IBM)-I(MM)) provides a good measure of the concentration of the hybridized material.

[00169] Nucleic Acid Samples

[00170] As is apparent to one of ordinary skill in the art, nucleic acid samples used in the methods and assays of the invention may be prepared by any available method or process. Methods of isolating total mRNA are also well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I Theory and Nucleic Acid Preparation, Tijssen, (1993) (editor) Elsevier Press. Such samples include RNA samples, but also include cDNA synthesized from an mRNA sample isolated from a cell or tissue of interest. Such samples also include DNA amplified from the cDNA, and an RNA transcribed from the amplified DNA. One of skill in the art would appreciate that it is desirable to inhibit or destroy RNase present in homogenates before homogenates can be used.

[00171] Biological samples may be of any biological tissue or fluid or cells from any organism as well as cells raised in vitro, such as cell lines and tissue culture cells. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Typical clinical samples include, but are not limited to, sputum, blood, blood-cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells.

[00172] Biological samples may also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological purposes.

[00173] High Density Arrays

[00174] Methods of forming high density arrays of oligonucleotides with a minimal number of synthetic steps are known. The oligonucleotide analogue array can be synthesized on a solid substrate by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling (see Pirrung et al., (1992) U.S. Pat. No. 5,143, 854; Fodor et al., (1998) U.S. Pat. No. 5,800,992; Chee et al, (1998) U.S. Pat. No. 5,837,832

[00175] In brief, the light-directed combinatorial synthesis of oligonucleotide arrays on a glass surface precedes using automated phosphoramidite chemistry and chip masking techniques. In one specific implementation, a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group. Photolysis through a photolithogaphic mask is used selectively to expose functional groups which are then ready to react with incoming 5' photoprotected nucleoside phosphoramidites. The phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group). Thus, the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences has been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents.

[00176] In addition to the foregoing, additional methods which can be used to generate an array of oligonucleotides on a single substrate are described in Fodor et al., (1993). WO 93/09668. High density nucleic acid arrays can also be fabricated by depositing premade or natural nucleic acids in predetermined positions. Synthesized or natural nucleic acids are deposited on specific locations of a substrate by light directed targeting and oligonucleotide directed targeting. Another embodiment uses a dispenser that moves from region to region to deposit nucleic acids in specific spots.

[00177] Hybridization

[00178] Nucleic acid hybridization simply involves contacting a probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing (see Lockhart et al., (1999) WO 99/32660). The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids.

[00179] Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA-DNA, RNA-RNA or RNA-DNA) will form even where the annealed sequences are not perfectly complementary.

[00180] Thus specificity of hybridization is reduced at lower stringency.

Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches. One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency, in this case in 6. times. S SPE-T at 37⁰C. (0.005% Triton x-100) to ensure hybridization and then subsequent washes are performed at higher stringency (e.g., 1. times. S SPE-T at 37⁰C.) to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25.times.SSPET at 37⁰C. to 50⁰C.) until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level controls, normalization controls, mismatch controls, etc.).

[00181] In general, there is a tradeoff between hybridization specificity

(stringency) and signal intensity. Thus, in a preferred embodiment, the wash is performed at the highest stringency that produces consistent results and that provides signal intensity greater than approximately 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest.

[00182] Signal Detection

[00183] The hybridized nucleic acids are typically detected by detecting one or more labels attached to the sample nucleic acids. The labels may be incoφorated by any of a number of means well known to those of skill in the art (see Lockhart et al., (1999) WO 99/32660).

[00184] Databases

[00185] The present invention includes relational databases containing sequence information, for instance for the genes of Tables 1-3, as well as gene expression information in various nerve tissue samples. Databases may also contain information associated with a given sequence or tissue sample such as descriptive information about the gene associated with the sequence information, or descriptive information concerning the clinical status of the tissue sample, or the patient from which the sample was derived. The database may be designed to include different parts, for instance a sequences database and a gene expression database. Methods for the configuration and construction of such databases are widely available, for instance, see Akerblom et al., (1999) U.S. Pat. No. 5,953,727, which is herein incorporated by reference in its entirety.

[00186] The databases of the invention may be linked to an outside or external database. In a preferred embodiment, the external database is GenBank and the associated databases maintained by the National Center for Biotechnology Information (NCBI).

[00187] Any appropriate computer platform may be used to perform the necessary comparisons between sequence information, gene expression information and any other information in the database or provided as an input. For example, a large number of computer workstations are available from a variety of manufacturers, such has those available from Silicon Graphics. Client-server environments, database servers and networks are also widely available and appropriate platforms for the databases of the invention. [00188] The databases of the invention may be used to produce, among other things, electronic Northerns to allow the user to determine the cell type or tissue in which a given gene is expressed and to allow determination of the abundance or expression level of a given gene in a particular tissue or cell.

[00189] The databases of the invention may also be used to present information identifying the expression level in a tissue or cell of a set of genes comprising at least one gene in Tables 1-3 comprising the step of comparing the expression level of at least one gene in Tables 1-3 in the tissue to the level of expression of the gene in the database. Such methods may be used to predict the physiological state of a given tissue by comparing the level of expression of a gene or genes in Tables 1-3 from a sample to the expression levels found in tissue from normal, malignant or neurofibromatosis and/or MPNST. Such methods may also be used in the drug or agent screening assays as described below.

Table 1. Group C9 genes showing increased expression in malignant tumors only.

Group c9

Gene

Systematic Common Symbol Description RefSeq septin 3 (SEPT3), transcript

NM_019106_at 3-Sep 3-Sep variant B, mRNA NM 019106 septin 3 (SEPT3), transcript

NM_145733_at 3-Sep 3-Sep variant A, mRNA NM 019106

ADAM metallopeptidase with thrombospondin type 1 motif,

NM_014243_at ADAMTS3 ADAMTS3 3 (ADAMTS3), mRNA NM 014243 adducin 2 (beta) (ADD2), transcript variant beta-1,

NM_001617_at ADD2 ADD2 mRNA NM 001617 adducin 2 (beta) (ADD2), transcript variant beta-3a,

NM 017483 at ADD2 ADD2 mRNA NM 001617 adducin 2 (beta) (ADD2), transcript variant beta-4,

NM_017488_at ADD2 ADD2 mRNA NM_001617 archaemetzincins-2 (AMZ2), NM_00103356

NM_001033569_at AMZ2 transcript variant 2, mRNA 9 archaemetzincins-2 ( AMZ2), NM_00103356

NMJ)01033570_at AMZ2 transcript variant 3, mRNA 9 archaemetzincins-2 (AMZ2), NM_00103356

NM_001033571_at AMZ2 transcript variant 4, mRNA 9 archaemetzincins-2 ( AMZ2), NMJ)0103356

NM_001033572_at AMZ2 transcript variant 5, mRNA 9 archaemetzincins-2 (AMZ2), NM 00103356

NM_001033574_at AMZ2 transcript variant 6, mRNA 9 archaemetzincins-2 (AMZ2), NM_00103356

NM_016627_at AMZ2 transcript variant 1, mRNA 9 amyloid beta (A4) precursor protein-binding, family A, member 2 (XI l -like)

NM_005503_at APBA2 APBA2 (APBA2), mRNA NM_005503

Rho GTPase activating protein

28 (ARHGAP28), transcript NM_00101000

NM_001010000_at ARHGAP28 ARHGAP28 variant 1, mRNA 0 astrotactin (ASTN), transcript

NM_004319_at ASTN ASTNl variant 1, mRNA NM 004319

BAIl -associated protein 2-like

NM_018842_at BAIAP2L1 BAIAP2L1 1 (BAI AP2L1), mRNA NMJ)18842 bagpipe homeobox homolog 1

NM_001189_at BAPXl BAPXl (Drosophila) (BAPXl), mRNA NM OOl 189 chromosome 14 open reading

NM 174978_at C14orβ9 C14orβ9 frame 39 (C14orf39), mRNA NM 174978 chromosome 20 open reading

NM_021215_at C20orf77 C20orf77 frame 77 (C20orf77), mRNA NM 021215 chromosome 20 open reading

NM_153269_at C20orf96 C20orf96 frame 96 (C20orf96), mRNA NMJ 53269 chromosome 6 open reading

NM 198920_at C6orΩ57 C6orfl57 frame 157 (C6orfl 57), mRNA NM 198920 chromosome 8 open reading

NM_032334_at C8orf53 C8orf53 frame 53 (C8orf53), mRNA NMJ)32334 chromosome 9 open reading

NM_032342_at C9orfl25 C9orfl25 frame 125 (C9orfl25), mRNA NMJ)32342

Cdk5 and AbI enzyme substrate 1 (CABLESl),

NM_138375_at CABLESl CABLESl mRNA NMJ38375 calcium channel, voltage- dependent, beta 2 subunit

(CACNB2), transcript variant NM_000724_at CACNB2 CACNB2 l, mRNA NMJJ00724 calcium channel, voltage- dependent, beta 2 subunit

(CACNB2), transcript variant NM 201570 at CACNB2 CACNB2 7, mRNA NMJ)00724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201571_at CACNB2 CACNB2 6, mRNA NM_ 000724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201572_at CACNB2 CACNB2 8, mRNA NM_ _.000724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201590_at CACNB2 CACNB2 3, mRNA NM_ _.000724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201593_at CACNB2 CACNB2 5, mRNA NM_ _000724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201596_at CACNB2 CACNB2 2, mRNA NM_ _000724 calcium channel, voltage- dependent, beta 2 subunit (CACNB2), transcript variant

NM_201597_at CACNB2 CACNB2 4, mRNA NM 000724 calmodulin-like 4 (CALML4), NDvf _00103173

NM_001031733_at CALML4 CALML4 transcript variant 1, mRNA 3 cartilage paired-class homeoprotein 1 (CARTl),

NM_006982_at CARTl CARTl mRNA NM_ _006982

CAZ-associated structural

NM_015576_at CASTl ERC2 protein (CASTl), mRNA NM_ J)15576 chromobox homolog 2 (Pc class homolog, Drosophila) (CBX2), transcript variant 1,

NM_005189_at CBX2 CBX2 mRNA NM_ _005189

CDC42 effector protein (Rho GTPase binding) 4

NM_012121_at CDC42EP4 CDC42EP4 (CDC42EP4), mRNA NM_ 012121 CKLF-like MARVEL transmembrane domain containing 7 (CMTM7),

NM_138410_at CMTM7 CMTM7 transcript variant 1, mRNA NM_ J38410 CKLF-like MARVEL transmembrane domain containing 7 (CMTM7),

NM_181472_at CMTM7 CMTM7 transcript variant 2, mRNA NM_. J38410 contactin associated protein 1

NM_003632_at CNTNAPl CNTNAPl (CNTNAPl), mRNA NM_. _003632 coagulation factor C homolog, cochlin (Limulus polyphemus)

NM 004086 at COCH COCH (COCH), mRNA NM 004086 collagen, type XIII, alpha 1

(COL13A1), transcript variant

NM_080804_at COL13A1 COL13A1 8, mRNA NM_005203 collagen, type XIII, alpha 1

(COL13A1), transcript variant

NM_080807_at COLl 3Al COL13A1 l l, mRNA NM_005203 collagen, type XIII, alpha 1

(COL13A1), transcript variant

NM_080808_at COL 13Al COL13A1 12, mRNA NM_005203 collagen, type XIII, alpha 1 (COLl 3Al), transcript variant

NM_080809_at COLl 3Al COL 13Al 13, mRNA NM_005203 collagen, type XIII, alpha 1 (COL 13Al), transcript variant

NM_080811_at COLl 3Al COL13A1 15, mRNA NM_005203 collagen, type IV, alpha 5 (Alport syndrome) (COL4A5),

NM_000495_at COL4A5 COL4A5 transcript variant 1, mRNA NM_000495 collagen, type IV, alpha 5 (Alport syndrome) (COL4A5),

NM_033380_at COL4A5 COL4A5 transcript variant 2, mRNA NM 000495 collagen, type IV, alpha 5 (Alport syndrome) (COL4A5),

NM_033381_at COL4A5 COL4A5 transcript variant 3, mRNA NM_000495 collagen, type IV, alpha 6 (COL4A6), transcript variant

NM_001847_at COL4A6 COL4A6 A, mRNA NMJ)01847 collagen, type IV, alpha 6 (COL4A6), transcript variant

NM_033641_at COL4A6 COL4A6 B, mRNA NM OO 1847 carboxypeptidase X (M 14

NM_019609_at CPXM CPXMl family) (CPXM), mRNA NM_019609 centrosome and spindle pole associated protein 1 (CSPPl),

NM_024790_at CSPPl CSPPl mRNA NM 024790 cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), nuclear gene encoding mitochondrial

NM_000782_at CYP24A1 CYP24A1 protein, mRNA NM_000782 dachsous 1 (Drosophila)

NM_003737_at DCHSl DCHSl (DCHSl), mRNA NM_003737 discs, large homolog 3 (neuroendocrine-dlg,

NM_021120_at DLG3 DLG3 Drosophila) (DLG3), mRNA NM_021120 delta-like 1 homolog (Drosophila) (DLKl), NM_00103299

NM_001032997_at DLKl DLKl transcript variant 2, mRNA 7 delta-like 1 homolog (Drosophila) (DLKl), NM_00103299

NM 003836 at DLKl DLKl transcript variant 1, mRNA 7 doublesex and mab-3 related transcription factor 1

NM_021951_at DMRTl DMRTl (DMRTl), mRNA NM 021951

PREDICTED: DMRT-like

XM_027162_at DMRTA2 DMRTA2 family A2 (DMRTA2), mRNA XM_027162

PREDICTED: DMRT-like

XM_941606_at DMRTA2 DMRTA2 family A2 (DMRTA2), mRNA XM_027162 delta-notch-like EGF repeat- containing transmembrane

NM_139072_at DNER DNER (DNER), mRNA NMJ39072

DNA (cytosine-5-)- methyltransferase 3 beta

(DNMT3B), transcript variant

NM_006892_at DNMT3B DNMT3B l. mRNA NM 006892

DNA (cytosine-5-)- methyltransferase 3 beta

(DNMT3B), transcript variant

NM_175848_at DNMT3B DNMT3B 2, mRNA NM 006892

DNA (cytosine-5-)- methyltransferase 3 beta

(DNMT3B), transcript variant

NM_175849_at DNMT3B DNMT3B 3, mRNA NM 006892

DNA (cytosine-5-)- methyltransferase 3 beta

(DNMT3B), transcript variant

NM_175850_at DNMT3B DNMT3B 6, mRNA NM_006892 dual specificity phosphatase 9

NM_001395_at DUSP9 DUSP9 (DUSP9), mRNA NM_001395

EF-hand calcium binding

NM_019065_at EFCBP2 EFCBP2 protein 2 (EFCBP2), mRNA NM O 19065 eukaryotic translation initiation factor 3, subunit 3 gamma,

NM_003756_at EIF3S3 EIF3S3 4OkDa (EIF3S3), mRNA NM_003756 engrailed homolog 2 (EN2),

NM_001427_at EN2 EN2 mRNA NM_001427

EPH receptor B2 (EPHB2),

NM_004442_at EPHB2 EPHB2 transcript variant 2, mRNA NM_004442

EPH receptor B2 (EPHB2),

NM_017449_at EPHB2 EPHB2 transcript variant 1, mRNA NM_004442

EPH receptor B4 (EPHB4),

NM_004444_at EPHB4 EPHB4 mRNA NM_004444 eyes absent homolog 4

(Drosophila) (EYA4),

NM_004100_at EYA4 EYA4 transcript variant 1, mRNA NM_004100 eyes absent homolog 4

(Drosophila) (EYA4),

NM_172103_at EYA4 EYA4 transcript variant 2, mRNA NM_004100 eyes absent homolog 4

(Drosophila) (EYA4),

NM_172105_at EYA4 EYA4 transcript variant 4, mRNA NM_004100

NM_001992_at F2R F2R coagulation factor II NM 001992 (thrombin) receptor (F2R), mRNA family with sequence similarity 119, member B

(FAMl 19B), transcript variant

NM_015433_at FAMl 19B FAMl 19B l, mRNA NM O 15433 family with sequence similarity 119, member B

(FAMl 19B), transcript variant

NM_206914_at FAMl 19B FAMl 19B 2, mRNA NMJ) 15433 family with sequence similarity 38, member B

NM_022068_at FAM38B FAM38B (FAM38B), mRNA NM 022068 hypothetical protein FLJ12505

NM_024749_at FLJ 12505 VASH2 (FLJ12505), mRNA NM_024749 hypothetical protein FLJ20130

NM_017681_at FLJ20130 NUP62CL (FLJ20130), mRNA NM_017681 forkhead box Al (FOXAl),

NM_004496_at FOXAl FOXAl mRNA NM_004496 forkhead box A2 (F0XA2),

NM_021784_at FOXA2 FOXA2 transcript variant 1 , mRNA NM 021784 forkhead box A2 (F0XA2),

NM_153675_at FOXA2 FOXA2 transcript variant 2, mRNA NM J)21784 forkhead box El (thyroid transcription factor 2)

NM_004473_at FOXEl FOXEl (FOXEl), mRNA NM_004473 forkhead box Fl (FOXFl),

NM_001451_at FOXFl FOXFl mRNA NMJ)01451 forkhead box GlB (FOXGlB),

NM_005249_at FOXGlB FOXGlB mRNA NMJ)05249 frataxin (FXN), nuclear gene encoding mitochondrial protein, transcript variant 1,

NM_000144_at FXN FXN mRNA NMJ)OO 144 frizzled homolog 2

NM 001466 at FZD2 FZD2 (Drosophila) (FZD2), mRNA NM OO 1466

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferas e 14 (GaINAc-TH)

NM 024572 at GALNT14 GALNT14 (GALNT14), mRNA NM_024572

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferas e 7 (GalNAc-T7) (GALNT7),

NM_017423_at GALNT7 GALNT7 mRNA NM O 17423 glucocorticoid induced

NMJ38426_at GLCCIl GLCCIl transcript 1 (GLCCI 1 ), mRNA NM l 38426

UDP-glucuronic acid

NMJ)15554_at GLCE GLCE epimerase (GLCE), mRNA NMJ) 15554

NM 000516 at GNAS GNAS GNAS complex locus NM 000516 (GNAS), transcript variant 1 , mRNA

GNAS complex locus

(GNAS), transcript variant 4,

NM_ 016592_at GNAS GNAS mRNA NM_000516

GNAS complex locus

(GNAS), transcript variant 3,

NM_ 080425_at GNAS GNAS mRNA NM_000516

GNAS complex locus

(GNAS), transcript variant 2,

NM_ 080426_at GNAS GNAS mRNA NM_000516

G protein-coupled receptor 125

NM_ 145290_at GPRl 25 GPRl 25 (GPR125), mRNA NMJ45290

PREDICTED: G protein-coupled receptor 125,

XM_ _940797_at GPR125 transcript variant 2 (GPR125), mRNA

PREDICTED: G protein-coupled receptor 125,

XM_ 944788_at GPR125 transcript variant 3 (GPR125), mRNA

PREDICTED: G protein-coupled receptor 125,

XM_ 94479 l_at GPRl 25 transcript variant 4 (GPR125), mRNA ^'

PREDICTED: G protein-coupled receptor 125,

XM_ 944795_at GPR125 transcript variant 5 (GPR 125), mRNA

PREDICTED: G protein-coupled receptor 125,

XM_ 944804_at GPR125 transcript variant 6 (GPR125), mRNA

NM_ _003918_at GYG2 GYG2 glycogenin 2 (GYG2), mRNA NM_003918

H2A histone family, member

NM_ 018649_at H2AFY2 H2AFY2 Y2 (H2AFY2), mRNA NM_018649 hepatocyte growth factor

(hepapoietin A; scatter factor)

(HGF), transcript variant 1,

NM_ 00060 l_at HGF HGF mRNA NM_000601 hepatocyte growth factor

(hepapoietin A; scatter factor)

(HGF), transcript variant 2,

NM_ 00101093 l_at HGF HGF mRNA NM_000601 hepatocyte growth factor

(hepapoietin A; scatter factor)

(HGF), transcript variant 3,

NM_ 001010932_at HGF HGF mRNA NM_000601 hepatocyte growth factor

(hepapoietin A; scatter factor)

(HGF), transcript variant 4,

NM_ _001010933_at HGF HGF mRNA NM_000601 hedgehog interacting protein

NM_ _022475_at HHIP HHIP (HHIP), mRNA NM_022475 histone l, H2bd

(HISTl H2BD), transcript

NM_ 138720_at HIST1H2BD HIST1H2BD variant 2, mRNA NM_021063 high mobility group AT-hook

2 (HMGA2), transcript variant

NM 003483 at HMGA2 HMGA2 l. mRNA NM_003483 high-mobility group box 2

NM_002129_at HMGB2 HMGB2 (HMGB2), mRNA NM 002129 homeobox (H6 family) 2

NM_005519_at HMX2 HMX2 (HMX2), mRNA NM 005519 homeobox A13 (HOXA 13),

NM_000522_at HOXA 13 HOXA 13 mRNA NM 000522 homeobox A3 (HOXA3),

NM_030661_at HOXA3 HOXA3 transcript variant 1 , mRNA NM 030661 homeobox A3 (HOXA3),

NM_153631_at HOXA3 HOXA3 transcript variant 2, mRNA NM 030661 homeobox A3 (HOXA3),

NM_153632_at HOXA3 HOXA3 transcript variant 3, mRNA NM 030661 homeobox B13 (HOXB 13),

NM_006361_at HOXB 13 HOXB 13 mRNA NM 006361 homeobox C13 (HOXC 13),

NM_017410_at HOXC 13 HOXC 13 mRNA NM 017410 heparan sulfate 6-0- sulfotransferase 2 (HS6ST2),

NM_147175_at HS6ST2 HS6ST2 mRNA NM 147175 insulin-like growth factor 2 mRNA binding protein 1

NM 006546 at IGF2BP1 IGF2BP1 (IGF2BP1), mRNA NM_ 006546 inositol hexaphosphate kinase

2 (IHPK2), transcript variant 3, NM _.00100590

NMJ)Ol 005910_at IHPK2 IHPK2 mRNA 9 inositol hexaphosphate kinase

2 (IHPK2), transcript variant 4, NM _.00100590

NM_001005911_at IHPK2 IHPK2 mRNA 9 inositol hexaphosphate kinase

2 (IHPK2), transcript variant 5, NM _.00100590

NMJ)Ol 005912_at IHPK2 IHPK2 mRNA 9 inositol hexaphosphate kinase

2 (IHPK2), transcript variant 6, NM 00100590

NM_001005913_at IHPK2 IHPK2 mRNA 9

ISL 1 transcription factor,

LIM/homeodomain, (islet- 1)

NM_002202_at ISLl ISLl (ISLl), mRNA NM_ _002202 isochorismatase domain

NM_016048_at ISOCl ISOCl containing 1 (ISOCl), mRNA NM_ _016048 myo-inositol 1 -phosphate synthase Al (ISYNAl),

NM_016368_at ISYNAl mRNA NM_ 016368 jumonji domain containing IB

NM 016604 at JMJDlB JMJDlB (JMJDlB), mRNA NM_ _016604

KH domain containing, RNA binding, signal transduction associated 1 (KHDRBSl),

NM_006559_at KHDRBSl KHDRBSl mRNA NM 006559

PREDICTED: KIAA0888 protein, transcript

XM_032571_at KIAA0888 variant 1 (KIAA0888), mRNA XM 943524 at KIAA0888 PREDICTED: KIAA0888 protein, transcript variant 2 (KIAA0888), mRNA

PREDICTED: KIAA0888 protein, transcript

XM_943531_at KIAA0888 variant 3 (KIAA0888), mRNA

KIAA1212 (KIAA1212),

NM_018084_at KIAA1212 KIAA1212 mRNA NM 018084

KIAA1467 (KIAA1467),

NM_020853_at KIAA 1467 KIAA 1467 mRNA NM 020853

PREDICTED: KIAA1549 protein, transcript variant 1

XM_371956_at KIAA 1549 (KIAA 1549), mRNA XM 371956

PREDICTED: KIAA 1549 protein, transcript variant 2

XM_379932_at KIAA 1549 (KIA A 1549), mRNA XM 371956

PREDICTED: KIAA 1549 protein, transcript

XM_935389_at KIAA 1549 variant 3 (KIAA 1549), mRNA

PREDICTED: KIAA 1549 protein, transcript

XM_935390_at KIAA 1549 variant 4 (KIAA 1549), mRNA

PREDICTED: KIAA 1549 protein, transcript

XM_936004_at KIAA 1549 variant 5 (KIAAl 549), mRNA

PREDICTED: KIAA 1549 protein, transcript variant 6

XM_941132_at KIAA 1549 (KIAA 1549), mRNA XM 371956

PREDICTED: KIAA1549 protein, transcript

XM_944983_at KIAA 1549 variant 7 (KIAA 1549), mRNA

KIAA1622 (KIAA1622),

NM_058237_at KIAA 1622 KIAA 1622 transcript variant 1, mRNA NM 020958

KIAA 1904 protein

NM_052906_at KIAA 1904 LRRC62 (KIAA 1904), mRNA NM 052906 lysosomal-associated membrane protein 3 (LAMP3),

NM_014398_at LAMP3 LAMP3 mRNA NM 014398 leucine zipper, down-regulated

NM_012317_at LDOCl LDOCl in cancer 1 (LDOC 1 ), mRNA NM_012317 leucine zipper-EF-hand containing transmembrane

NM_144652_at LETM2 LETM2 protein 2 (LETM2), mRNA NM 144652 lin-28 homolog B (C. elegans) NM^~ [00100431

NM_001004317_at LIN28B LIN28B (LIN28B), mRNA 7 lipase, endothelial (LIPG),

NM 006033 at LIPG LIPG mRNA NM_ _006033

PREDICTED: hypothetical gene supported by BX248251

XM 370758 at LOC387978 (LOC387978), mRNA XM_ _370758

PREDICTED: hypothetical gene supported by BX248251

XM_940323_at LOC387978 (LOC387978), mRNA XM _370758

PREDICTED: hypothetical gene supported by AK 124295

XM_374260_at LOC389641 (LOC389641), mRNA XM _374260

PREDICTED: hypothetical XM 944097 at LOC389641 gene supported by AK 124295 XM 374260 (LOC389641 ), mRNA

PREDICTED: hypothetical protein LOC650494

XM 944224 at LOC650494 (LOC650494), mRNA PREDICTED: hypothetical protein LOC92312

XM 044166 at LOC92312 (LOC92312), mRNA XM_044166

PREDICTED: hypothetical protein LOC92312

XM_937993_at LOC92312 (LOC92312), mRNA XM_044166 latrophilin 1 (LPHNl), NM_00100870

NM_001008701_at LPHNl LPHNl transcript variant 1, mRNA 1 latrophilin 1 (LPHN 1 ), NM_00100870

NM_014921_at LPHNl LPHNl transcript variant 2, mRNA 1 mitogen-activated protein

NM 002758 at MAP2K6 MAP2K6 kinase 6 (MAP2K6), mRNA NM 002758 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) associated protein

MCM3APA MCM3APA antisense (MCM3 APAS) on

NR_002776_at S S chromosome 21 NR 002776 mucolipin 3 (MCOLN3), NM 018298 at MCOLN3 MCOLN3 mRNA NM O 18298 midkine (neurite growth- promoting factor 2) (MDK), NM_00101233

NM_001012333_at MDK MDK transcript variant 2, mRNA 3 midkine (neurite growth- promoting factor 2) (MDK), NM OO 101233

NM_001012334_at MDK MDK transcript variant 1, mRNA 3 midkine (neurite growth- promoting factor 2) (MDK), NM_00101233

NM_002391_at MDK MDK transcript variant 3, mRNA 3

PREDICTED: hypothetical protein MGC 18216

XM_927732_at MGC 18216 (MGC 18216), mRNA

PREDICTED: hypothetical protein MGC18216

XM 939617 at MGC18216 (MGC 18216), mRNA mitochondrial ribosomal protein L32 (MRPL32), nuclear gene encoding

NM_031903_at MRPL32 MRPL32 mitochondrial protein, mRNA NM_031903 msh homeobox homolog 2

NM_002449_at MSX2 MSX2 (Drosophila) (MSX2), mRNA NM_002449 MTERF domain containing 2

NM_182501_at MTERFD2 MTERFD2 (MTERFD2), mRNA NM_182501 myotubularin related protein 4

NM_004687_at MTMR4 MTMR4 (MTMR4), mRNA NM_004687 melanoma associated antigen

NM_032853_at MUMl MUMl (mutated) 1 (MUMl), mRNA NM 032853 nuclear casein kinase and cyclin-dependent kinase

NM 022731 at NUCKSl NUCKSl substrate 1 (NUCKSl), mRNA NM_022731 purinergic receptor P2X, ligand-gated ion channel, 5

(P2RX5), transcript variant 1,

NM 002561 at P2RX5 P2RX5 mRNA NM_ 002561 purinergic receptor P2X, ligand-gated ion channel, 5

(P2RX5), transcript variant 2,

NM 175080 at P2RX5 P2RX5 mRNA NM_ 002561 poly(A) binding protein, cytoplasmic 1 (PABPCl),

NM 002568 at PABPCl PABPCl mRNA NM_ _002568 paired box gene 6 (aniridia, keratitis) (PAX6), transcript

NM 000280 at PAX6 PAX6 variant 1, mRNA NM_ _.000280 paired box gene 6 (aniridia, keratitis) (PAX6), transcript

NM 001604 at PAX6 PAX6 variant 2, mRNA NM_ 000280 phosphodiesterase 7A

(PDE7A), transcript variant 1,

NM 002603 at PDE7A PDE7A mRNA NM_ _.002603 phosphodiesterase 7A

(PDE7A), transcript variant 2,

NM 002604 at PDE7A PDE7A mRNA NM_ _002603

PHD finger protein 14

(PHF 14), transcript variant 1, NM J)0100715

NM 001007157 at PHF14 PHF14 mRNA 7

PHD finger protein 14

(PHF 14), transcript variant 2, NM _00100715

NM 014660 at PHF14 PHF14 mRNA 7 phospholipase C, gamma 1

(PLCGl), transcript variant 1,

NM 002660 at PLCGl PLCGl mRNA NM_{. „}002660 phospholipase C, gamma 1

(PLCGl), transcript variant 2,

NM 182811 at PLCGl PLCGl mRNA NM_ _002660 phorbol- 12-myristate- 13- acetate-induced protein 1

NM 021127 at PMAIPl PMAIP 1 (PMAIP 1 ), mRNA NM_. 021127

POTl protection of telomeres

1 homolog (S. pombe) (POTl), NM _00104259

NM 015450 at POTl POTl mRNA 4

POU domain, class 4, transcription factor 1

NM_006237_at POU4F1 POU4F 1 (POU4F 1 ), mRNA NM _006237 protein phosphatase IE (PP2C domain containing) (PPMlE),

NM_014906_at PPMlE PPMlE mRNA NM_. _014906

PR domain containing 13

NM_021620_at PRDMl 3 PRDM13 (PRDM13), mRNA NM 021620 NM 006017 at PROMl PROMl prominin 1 (PROMl), mRNA NM 006017 NM_181716_at PRR6 PRR6 proline rich 6 (PRR6), mRNA NMJ81716 protease, serine, 16 (thymus)

NM_005865_at PRSS 16 PRSS 16 (PRSS 16), mRNA NM_005865

PC4 and SFRSl interacting protein 1 (PSIPl), transcript

NM_021144_at PSIPl PSIPl variant l, mRNA NM_021144

PC4 and SFRSl interacting protein 1 (PSIPl), transcript

NM_033222_at PSIPl PSIPl variant 2, mRNA NM_021144 protein tyrosine phosphatase type IVA, member 3

(PTP4A3), transcript variant 2,

NM_007079_at PTP4A3 PTP4A3 mRNA NM_007079 protein tyrosine phosphatase type IVA, member 3

(PTP4A3), transcript variant 1,

NM_032611_at PTP4A3 PTP4A3 mRNA NM_007079 protein tyrosine phosphatase, receptor type, D (PTPRD), NM 00104071

NMJ)Ol 040712_at PTPRD PTPRD transcript variant 5, mRNA 2 protein tyrosine phosphatase, receptor type, D (PTPRD), NM 00104071

NM_002839_at PTPRD PTPRD transcript variant 1, mRNA 2 protein tyrosine phosphatase, receptor type, D (PTPRD), NM 00104071

NM_130391_at PTPRD PTPRD transcript variant 2, mRNA 2 protein tyrosine phosphatase, receptor type, D (PTPRD), NM 00104071

NM_130392_at PTPRD PTPRD transcript variant 3, mRNA 2 protein tyrosine phosphatase, receptor type, D (PTPRD), NM 00104071

NM 130393 at PTPRD PTPRD transcript variant 4, mRNA 2 quinolinate phosphoribosyltransferase

(nicotinate-nucleotide pyrophosphorylase

(carboxylating)) (QPRT),

NM_014298_at QPRT QPRT mRNA NM_014298

PREDICTED: RNA binding motif protein 20 (RBM20),

XM_291671_at RBM20 RBM20 mRNA XM 291671

PREDICTED: RNA binding motif protein 20 (RBM20),

XM_939337_at RBM20 RBM20 mRNA XM_291671 regulator of G-protein

NM_012419_at RGS 17 RGS 17 signalling 17 (RGS 17), mRNA NMJ)12419 ring finger protein 175

NM_173662_at RNF175 RNF 175 (RNF 175), mRNA NM_173662 ribosomal protein S6 kinase,

7OkDa, polypeptide 1

NM 003161 at RPS6KB1 RPS6KB1 (RPS6KB1), mRNA NM 003161 SlOOP binding protein

(SlOOPBP), transcript variant NM_00101740

NM_001017406_at SlOOPBP SlOOPBP 2, mRNA 6

SEC14-like 1 (S. cerevisiae)

(SEC 14Ll), transcript variant NMJ)0103957

NMJ)01039573_at SEC14L1 SEC14L1 2, mRNA 3 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)

NM_006080_at SEMA3A SEMA3A 3A (SEMA3A), mRNA NMJ)06080 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B

(SEMA6B), transcript variant

NM_020241_at SEMA6B SEMA6B SEMA6B.l, mRNA NMJ)20241 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B

(SEMA6B), transcript variant

NM_032108_at SEMA6B SEMA6B SEMA6B.3, mRNA NMJ)20241 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B

(SEMA6B), transcript variant

NM_133327_at SEMA6B SEMA6B SEMA6B.2, mRNA NMJ)20241

SIN3 homolog A, transcription regulator (yeast) (SIN3A),

NM_015477_at SIN3A SIN3A mRNA NMJ) 15477 sine oculis homeobox homolog

NM_005982_at SIXl SIXl 1 (Drosophila) (SIXl), mRNA NMJ)05982

S-phase kinase-associated protein 2 (p45) (SKP2),

NM_032637_at SKP2 SKP2 transcript variant 2, mRNA NM 005983 solute carrier family 16

(monocarboxylic acid transporters), member 9

NM_194298_at SLC16A9 SLC16A9 (SLC 16 A9), mRNA NM_194298 solute carrier family 22

(organic cation transporter),

NM_003060_at SLC22A5 SLC22A5 member 5 (SLC22A5), mRNA NMJJ03060 solute carrier family 35, member F3 (SLC35F3),

NM_173508_at SLC35F3 SLC35F3 mRNA NM_173508 solute carrier family 39 (metal ion transporter), member 11

NM_139177_at SLC39A11 SLC39A11 (SLC39Al l), mRNA NM 139177

SMAD, mothers against DPP homolog 3 (Drosophila)

NM_005902_at SMAD3 SMAD3 (SMAD3), mRNA NMJ)05902 synuclein, alpha interacting

NM 005460 at SNCAIP SNCAIP protein (synphilin) (SNCAIP), NM 005460 mRNA small nucleolar RNA, H/ACA box 72 (SNORA72) on

NR_002581_at SNORA72 SNORA72 chromosome 8 NR_002581

SRY (sex determining region

NM_003108_at SOXI l SOXI l Y)-box 11 (SOXI l ), mRNA NM 003108

PREDICTED: U2-associated

SR140 protein (SR140),

XM_031553_at SRl 40 mRNA XM_031553

PREDICTED: U2-associated

SR140 protein (SR140),

XM_940802_at SR140 mRNA XM_031553 single stranded DNA binding protein 4 (SSBP4), transcript NM 00100999

NM_001009998_at SSBP4 SSBP4 variant 2, mRNA 8 single stranded DNA binding protein 4 (SSBP4), transcript NM 00100999

NM_032627_at SSBP4 SSBP4 variant l, mRNA 8 stathmin-like 3 (STMN3),

NM_015894_at STMN3 STMN3 mRNA NMJ)15894 suppressor of hairy wing homolog 3 (Drosophila)

NM_017666_at SUH W3 SUHW3 (SUHW3), mRNA NM_017666 transcriptional adaptor 1 (HFIl homolog, yeast)-like

NM_053053_at TADAlL TADAlL (TADAlL), mRNA NM_053053 transcription elongation factor

A (SΙI)-like 2 (TCEAL2),

NM_080390_at TCEAL2 TCEAL2 mRNA NM_080390 transcription factor 20 (ARl)

(TCF20), transcript variant 1,

NM_005650_at TCF20 TCF20 mRNA NM_005650 transcription factor 20 (ARl)

(TCF20), transcript variant 2,

NM_181492_at TCF20 TCF20 mRNA NM_005650 transmembrane protein with

EGF-like and two follistatin- like domains 2 (TMEFF2),

NM_016192_at TMEFF2 TMEFF2 mRNA NM Ol 6192 transmembrane protein 156

NM_024943_at TMEM 156 TMEM 156 (TMEMl 56), mRNA NM 024943 transmembrane protein 46 NM 00100753

NMJ⁾Ol 007538_at TMEM46 TMEM46 (TMEM46), mRNA 8 troponin T type 1 (skeletal,

NM_003283_at TNNTl TNNTl slow) (TNNTl), mRNA NM_003283 thyrotropin-releasing hormone degrading enzyme (TRHDE),

NM_013381_at TRHDE TRHDE mRNA NM_013381 tripartite motif-containing 37

(TRIM37), transcript variant 2, NM 00100520

NM 001005207 at TRIM37 TRIM37 mRNA 7 transient receptor potential cation channel, subfamily A,

NM_007332_at TRPAl TRPAl member 1 (TRPAl), mRNA NM 007332 transient receptor potential cation channel, subfamily C,

NM_003305_at TRPC3 TRPC3 member 3 (TRPC3), mRNA NM_003305 transformation/transcription domain-associated protein

NM_003496_at TRRAP TRRAP (TRRAP), mRNA NM_003496

26S proteasome-associated UCH interacting

NM_207106_at UIPl protein 1 (UIPl), transcript variant 3, mRNA unc-84 homolog A (C.

NM_025154_at UNC84A UNC84A elegans) (UNC84 A), mRNA NM_025154 vestigial like 2 (Drosophila) (VGLL2), transcript variant 2,

NM_153453_at VGLL2 VGLL2 mRNA NM_153453 vestigial like 2 (Drosophila) (VGLL2), transcript variant 1,

NM_182645_at VGLL2 VGLL2 mRNA NM_153453

WEEl homolog (S. pombe)

NM_003390_at WEEl WEEl (WEEl), mRNA NM_003390

Wilms tumor 1 (WTl),

NM_000378_at WTl WTl transcript variant A, mRNA NM_000378 Wilms tumor 1 (WTl),

NM_024424_at WTl WTl transcript variant B, mRNA NM 000378 Wilms tumor 1 (WTl),

NM_024425_at WTl WTl transcript variant C, mRNA NM 000378 Wilms tumor 1 (WTl),

NM_024426_at WTl WTl transcript variant D, mRNA NM_000378 X-linked Kx blood group (McLeod syndrome) (XK),

NM_021083_at XK XK mRNA NM_021083 zinc finger, CCHC domain containing 3 (ZCCHC3),

NM_033089_at ZCCHC3 ZCCHC3 mRNA NM_033089 zinc finger, DHHC-type containing 23 (ZDHHC23),

NM_173570_at ZDHHC23 ZDHHC23 mRNA NM_173570 zinc finger, ANl -type domain

NM_024699_at ZFANDl ZFANDl 1 (ZFANDl), mRNA NM_024699

Zic family member 1 (odd- paired homolog, Drosophila)

NM_003412_at ZICl ZICl (ZICl), mRNA NM 003412

Zic family member 2 (odd- paired homolog, Drosophila)

NM_007129_at ZIC2 ZIC2 (ZIC2), mRNA NM_007129 zinc finger protein 337

NM 015655 at ZNF337 ZNF337 (ZNF337), mRNA NM_015655 Table 2. Cluster 10 genes showing increased expression in benign and malignant tumors. nfl custom cdf (86) Group clO (batchesl+2+3)

Gene Systematic Common Symbol Description RefSeq acyl-CoA synthetase short-chain family member 2 (ACSS2), transcript variant 1, NM_018677_at ACSS2 ACSS2 mRNA NM_018677 acyl-CoA synthetase short-chain family member 2 (ACSS2), transcript variant 2, NM_139274_at ACSS2 ACSS2 mRNA NMJ)18677 amine oxidase, copper containing 3

(vascular adhesion protein 1) (AOC3), NM_003734_at AOC3 AOC3 mRNA NM_003734 apolipoprotein D NMJ)01647_at APOD APOD (APOD), mRNA NMJ)01647 aquaporin 1 (Colton blood group) NM_198098_at AQPl AQPl (AQPl), mRNA NM 198098

Rho GTPase activating protein 6

(ARHGAP6), transcript variant 2, NMJ)Ol 174_at ARHGAP6 ARHGAP6 mRNA NM OOl 174

Rho GTPase activating protein 6

(ARHGAP6), transcript variant 4, NMJ)13423_at ARHGAP6 ARHGAP6 mRNA NMJ)01174

Rho GTPase activating protein 6

(ARHGAP6), transcript variant 1, NM_013427_at ARHGAP6 ARHG AP6 mRNA NMJ)01174

Rho GDP dissociation inhibitor

(GDI) beta NM OOl 175_at ARHGDIB ARHGDIB (ARHGDIB), mRNA NM OOl 175 chromosome 2 open reading frame 32 NMJ)15463_at C2orf32 C2orf32 (C2orf32), mRNA NMJ) 15463 chromosome 6 open reading frame 192

NM 052831 at C6orfl92 C6orfl92 (C6orfl92), mRNA NM_052831 chromosome 6 open reading frame 32

NM 015864 at C6orf32 C6orf32 (C6orfi2), mRNA NMJ)15864 chromosome 9 open reading frame 26

(NF-HEV)

NM 033439 at C9orf26 C9orf26 (C9orf26), mRNA NM_033439 cache domain containing 1

NM 020925 at CACHDl CACHDl (CACHDl), mRNA NM_020925

Ca2+-dependent activator protein for secretion 2

(CADPS2), transcript

NM 001009571 at CADPS2 CADPS2 variant 2, mRNA NMJ)01009571

Ca2+-dependent activator protein for secretion 2

(CADPS2), transcript

NM 017954 at CADPS2 CADPS2 variant l, mRNA NMJ)01009571 caspase 1, apoptosis- related cysteine peptidase

(interleukin 1, beta, convertase)

(CASPl), transcript variant epsilon,

NM 033295 at CASPl CASPl mRNA NMJ)01223

CD36 molecule

(thrombospondin receptor) (CD36), transcript variant 3,

NM 000072 at CD36 CD36 mRNA NMJ)00072

CD36 molecule

(thrombospondin receptor) (CD36), transcript variant 2,

NM 001001547 at CD36 CD36 mRNA NMJ)00072

CD36 molecule

(thrombospondin receptor) (CD36), transcript variant 1,

NM 001001548 at CD36 CD36 mRNA NM 000072 collectin sub-family member 12

(COLEC12), transcript variant II,

NM 030781 at COLEC12 COLEC 12 mRNA NM 030781 collectin sub-family member 12

(COLEC12), transcript variant I,

NM_130386_at COLEC 12 COLEC 12 mRNA NM_030781 carboxypeptidase E

NMJ)01873_at CPE CPE (CPE), mRNA NMJ)01873 carboxypeptidase X

(M 14 family), member 2 (CPXM2),

NM_198148_at CPXM2 CPXM2 mRNA NM_198148 cathepsin K

(pycnodysostosis)

NM_000396_at CTSK CTSK (CTSK), mRNA NMJ)00396 cortactin binding protein 2

NMJ⁾33427_at CTTNBP2 CTTNBP2 (CTTNBP2), mRNA NM_033427 death-associated protein kinase 1

NM_004938_at DAPKl DAPKl (DAPKl), mRNA NM_004938 doublecortin and

CaM kinase-like 1

(DCAMKLl),

NM_004734_at DCAMKLl DCAMKLl mRNA NM 004734 development and differentiation enhancing factor-like

NM_017707_at DDEFLl DDEFLl 1 (DDEFLl), mRNA NMJ) 17707 dihydropyrimidinase- like 2 (DPYSL2),

NM_001386_at DPYSL2 DPYSL2 mRNA NM OO 1386

Down syndrome critical region gene

1-like 1 (DSCRlLl),

NM_005822_at DSCRlLl DSCRlLl mRNA NM_005822

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMPl), transcript variant 2,

NM_001039348_at EFEMPl EFEMPl mRNA NM OO 1039348

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMPl), transcript variant 3,

NMJ)01039349_at EFEMPl EFEMPl mRNA NM_001039348

EGF-containing fibulin-like extracellular matrix

NM 004105 at EFEMPl EFEMPl protein 1 (EFEMPl), NM 001039348 transcript variant 1, mRNA epidermal growth factor receptor

(erythroblastic leukemia viral (v- erb-b) oncogene homolog, avian)

(EGFR), transcript

NM 005228 at EGFR EGFR variant 1, mRNA NM_005228 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian)

NM 005239 at ETS2 ETS2 (ETS2), mRNA NM_005239 fetal Alzheimer antigen (FALZ), transcript variant 2,

NM 004459 at FALZ FALZ mRNA NM 004459 fetal Alzheimer antigen (FALZ), transcript variant 1 ,

NM 182641 at FALZ FALZ mRNA NM 004459 fibulin 1 (FBLNl), transcript variant D,

NM 006486 at FBLNl FBLNl mRNA NM 001996

Fc fragment of IgG, receptor, transporter, alpha (FCGRT),

NM 004107 at FCGRT FCGRT mRNA NM_004107

FCH and double SH3 domains 2

NM 014824 at FCHSD2 FCHSD2 (FCHSD2), mRNA NM_014824 fibroblast growth factor receptor 1

(fms-related tyrosine kinase 2, Pfeiffer syndrome) (FGFRl), transcript variant 2,

NM 015850 at FGFRl FGFRl mRNA NM_015850 fibroblast growth factor receptor 1

(fms-related tyrosine kinase 2, Pfeiffer syndrome) (FGFRl), transcript variant 3,

NM 023105 at FGFRl FGFRl mRNA NM 015850 fibroblast growth factor receptor 1

(fms-related tyrosine kinase 2, Pfeiffer

NM 023106 at FGFRl FGFRl syndrome) (FGFRl), NM_015850 transcript variant 4, mRNA fibroblast growth factor receptor 1

(fms-related tyrosine kinase 2, Pfeiffer syndrome) (FGFRl), transcript variant 1,

NM 023110 at FGFRl FGFRl mRNA NM_015850 fibroblast growth factor receptor 1

(fms-related tyrosine kinase 2, Pfeiffer syndrome) (FGFRl), transcript variant 9,

NM 023111 at FGFRl FGFRl mRNA NM_015850 four and a half LIM domains 1 (FHLl),

NM 001449 at FHLl FHLl mRNA NM_001449 hypothetical protein

FLJ10159

NM 018013 at FLJ10159 (FLJ10159), mRNA NMJH8013 hypothetical protein

FLJ23191

NM 024574 at FLJ23191 C4orf31 (FLJ23191), mRNA NM_024574

B cell RAG associated protein

(GALNAC4S-6ST),

NM 015892 at GALNAC4S-6ST mRNA NM_015892 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1

NM_002069_at GNAIl GNAIl (GNAIl), mRNA NM_002069 hemicentin 1 NM_031935_at HMCNl HMCNl (HMCNl), mRNA NM 031935 immediate early response 2 (IER2),

NM_004907_at IER2 IER2 mRNA NM_004907 immediate early response 5 (IER5), NM 016545 at IER5 IER5 mRNA NM_016545 immunoglobulin superfamily containing leucine- rich repeat (ISLR), transcript variant 1,

NM_005545_at ISLR ISLR mRNA NM_005545 immunoglobulin NM 201526 at ISLR ISLR superfamily NM 005545 containing leucine- rich repeat (ISLR), transcript variant 2, mRNA potassium inwardly- rectifying channel, subfamily J, member

NM_000891_at KCNJ2 KCNJ2 2 (KCNJ2), mRNA NM_000891 potassium channel, subfamily K, member 1 (KCNKl),

NM_002245_at KCNKl KCNKl mRNA NM_002245 v-kit Hardy- Zuckerman 4 feline sarcoma viral oncogene homolog

NM_000222_at KIT KIT (KIT), mRNA NM_000222

LIM domain binding

NM_001290_at LDB2 LDB2 2 (LDB2), mRNA NMJ)01290 leptin receptor (LEPR), transcript

NM_001003679_at LEPR LEPR variant 2, mRNA NM_001003679 leptin receptor (LEPR), transcript

NM_001003680_at LEPR LEPR variant 3, mRNA NM 001003679

PREDICTED: similar to matrilin 2 precursor, transcript variant 1

XM_209824_at LOC285929 (LOC285929), mRNA PREDICTED: similar to matrilin 2 precursor, transcript variant 2

XM_374399_at LOC285929 (LOC285929), mRNA PREDICTED: similar to matrilin 2 precursor, transcript variant 3

XM_932509_at LOC285929 (LOC285929), mRNA PREDICTED: similar to matrilin 2 precursor, transcript variant 4

XM_935894_at LOC285929 (LOC285929), mRNA PREDICTED: similar to matrilin 2 precursor, transcript variant 5

XM_936837_at LOC285929 (LOC285929), mRNA PREDICTED: similar to matrilin 2 precursor, transcript variant 6

XM_943018_at LOC285929 (LOC285929), mRNA PREDICTED: similar to sprouty homolog 4 (Drosophila) (LOC653170),

XM_926321_at LOC653170 mRNA

PREDICTED: similar to ARG99 protein

XM_928461_at LOC653626 (LOC653626), mRNA PREDICTED: similar to sprouty homolog 4 (Drosophila) (LOC654129),

XM 939732 at LOC654129 mRNA plasticity related gene 1 (LPPR4),

NM_014839_at LPPR4 mRNA NM_014839 leucine rich repeat containing 54

NM_015516_at LRRC54 LRRC54 (LRRC54), mRNA NMJ)15516 leucine rich repeat containing 8 family, member C

NM_032270_at LRRC8C LRRC8C (LRRC8C), mRNA NM_032270 lumican (LUM),

NM_002345_at LUM LUM mRNA NM_002345 mitogen-activated protein kinase kinase kinase 5 (MAP3K5),

NM_005923_at MAP3K5 MAP3K5 mRNA NM_005923 microfibrillar associated protein 5

NM 003480 at MFAP5 MFAP5 (MF AP5), mRNA NM_003480 membrane metallo- endopeptidase

(neutral endopeptidase, enkephalinase,

CALLA, CDlO)

(MME), transcript

NM 000902 at MME MME variant 1, mRNA NM 000902 membrane metallo- endopeptidase

(neutral endopeptidase, enkephalinase,

CALLA, CDlO)

(MME), transcript

NM 007287 at MME MME variant Ibis, mRNA NM 000902 membrane metallo- endopeptidase

(neutral endopeptidase, enkephalinase,

CALLA, CDlO)

(MME), transcript

NM 007288 at MME MME variant 2a, mRNA NM 000902 membrane metallo- endopeptidase

(neutral endopeptidase, enkephalinase,

CALLA, CDlO)

(MME), transcript

NM 007289 at MME MME variant 2b, mRNA NM_000902 matrix-remodelling associated 5

NM_015419_at MXRA5 MXRA5 (MXRA5), mRNA NM_015419 nuclear factor I/A

NM_005595_at NFIA NFIA (NFIA), mRNA NM_005595 nuclear factor I/B

NM_005596_at NFIB NFIB (NFIB), mRNA NM_005596 noggin (NOG),

NM_005450_at NOG NOG mRNA NM_005450 likely ortholog of mouse neighbor of

Punc El 1 (NOPE),

NM_020962_at NOPE mRNA NM 020962 odd-skipped related 1

(Drosophila)

NM_145260_at OSRl OSRl (OSRl), mRNA NMJ45260 phosphodiesterase

5A, cGMP-specific

(PDE5A), transcript

NMJ)01083_at PDE5A PDE5A variant 1, mRNA NM OO 1083 phosphodiesterase

5A, cGMP-specific

(PDE5A), transcript

NM_033437_at PDE5A PDE5A variant 3, mRNA NMJ)01083 podocalyxin-like

(PODXL), transcript

NM_001018111_at PODXL PODXL variant l, mRNA NM_001018111 podocalyxin-like

(PODXL), transcript

NM_005397_at PODXL PODXL variant 2, mRNA NMJ)01018111

Ras association

(RalGDS/AF-6) domain family 2

(RASSF2), transcript

NM_014737_at RASSF2 RASSF2 variant l. mRNA NM_014737

Ras association

(RalGDS/AF-6) domain family 2

(RASSF2), transcript

NM 170774_at RASSF2 RASSF2 variant 2, mRNA NMJ⁾14737 regulator of G- protein signalling 16

NM_002928_at RGS 16 RGS 16 (RGS 16), mRNA NM 002928 ribonuclease T2

NM_003730_at RNASET2 RNASET2 (RNASET2), mRNA NM_003730

RAR-related orphan receptor A (RORA), transcript variant 3,

NM_002943_at RORA RORA mRNA NM_002943

RAR-related orphan

NM_134260_at RORA RORA receptor A (RORA), NM 002943 transcript variant 2, mRNA

RAR-related orphan receptor A (RORA), transcript variant 1 ,

NM 134261 at RORA RORA mRNA NM 002943

RAR-related orphan receptor A (RORA), transcript variant 4,

NM 134262 at RORA RORA mRNA NM 002943 SlOO calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog) (S 100 A4), transcript

NM 002961 at S100A4 S100A4 variant l, mRNA NM 002961 SlOO calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog) (S 100 A4), transcript

NMJ)19554_at S100A4 S100A4 variant 2, mRNA NM 002961 Salvador homolog 1 (Drosophila)

NM_021818_at SAVl SAVl (SAVl), mRNA NM_021818 SET binding protein NM_015559_at SETBPl SETBPl 1 (SETBPl), mRNA NM O 15559 short stature homeobox 2 (SHOX2), transcript variant SHOX2b,

NM_003030_at SHOX2 SHOX2 mRNA NM_OO3O3O short stature homeobox 2 (SHOX2), transcript variant SHOX2a, NM_006884_at SHOX2 SHOX2 mRNA NM 003030 solute carrier family 1 (glial high affinity glutamate transporter), member NM_004172_at SLC1A3 SLCl A3 3 (SLCl A3), mRNA NM 004172 SWI/SNF related, matrix associated, actin dependent regulator of NM 003077 at SMARCD2 SMARCD2 chromatin, subfamily NM 003077 d, member 2

(SMARCD2), mRNA suppressor of cytokine signaling 3

NM 003955 at SOCS3 SOCS3 (SOCS3), mRNA NM_003955 sorbin and SH3 domain containing 2

(SORBS2), transcript

NM 003603 at SORBS2 SORBS2 variant 1, mRNA NM_003603 sorbin and SH3 domain containing 2

(SORBS2), transcript

NM 021069 at SORBS2 SORBS2 variant 2, mRNA NM_003603 sprouty homolog 1 , antagonist of FGF signaling

(Drosophila)

(SPRYl), transcript

NM 005841 at SPRYl SPRYl variant 1, mRNA NM_005841 sprouty homolog 1, antagonist of FGF signaling

(Drosophila)

(SPRYl), transcript

NM_199327_at SPRYl SPRYl variant 2, mRNA NM_005841 sprouty homolog 4

(Drosophila)

NM_030964_at SPRY4 SPRY4 (SPRY4), mRNA NM_030964 signal transducer and activator of transcription 5B

NM_012448_at STAT5B STAT5B (STAT5B), mRNA NM O 12448 stomatin (STOM), transcript variant 1,

NM_004099_at STOM STOM mRNA NM_004099 stomatin (STOM), transcript variant 2,

NM_198194_at STOM STOM mRNA NM 004099

SWAP-70 protein

NM_015055_at SWAP70 (SWAP70), mRNA NM_015055

TBCl domain family, member 4

NM_014832_at TBC1D4 TBC1D4 (TBClD4), mRNA NM_014832 transcription factor 8

(represses interleukin

2 expression)

NM_030751_at TCF8 TCF8 (TCF8), mRNA NM_030751 transmembrane and tetratricopeptide

NM 175861 at TMTCl TMTCl repeat containing 1 NM_175861 (TMTCl), mRNA tumor necrosis factor, alpha-induced protein 2

NM 006291 at TNFAIP2 TNFAIP2 (TNFAIP2), mRNA NM 006291 troponin T type 2 (cardiac) (TNNT2), transcript variant 1,

NM 000364 at TNNT2 TNNT2 mRNA NM 000364 troponin T type 2 (cardiac) (TNNT2), transcript variant 2,

NM 001001430 at TNNT2 TNNT2 mRNA NM 000364 troponin T type 2 (cardiac) (TNNT2), transcript variant 3,

NM 001001431 at TNNT2 TNNT2 mRNA NM 000364 troponin T type 2 (cardiac) (TNNT2), transcript variant 4,

NM 001001432 at TNNT2 TNNT2 mRNA NM 000364 tribbles homolog 1 (Drosophila)

NM 025195 at TRIBl TRIBl (TRIBl), mRNA NM 025195 zinc finger protein, multitype 2

NM 012082 at ZFPM2 ZFPM2 (ZFPM2), mRNA NM 012082 zinc finger protein 521 (ZNF521),

NM 015461 at ZNF521 ZNF521 mRNA NM 015461

Table 3. Cluster 11 genes showing increased expression in benign and malignant tumors.

Group ell nfl custom cdf (86) (batchesl+2+3)

Gene

Systematic Common Symbol Description RefSeq

PREDICTED: septin 8, transcript variant 1

XM_034872_at 8-Sep (SEPT8), mRNA

PREDICTED: septin 8, transcript variant 6

XM_943986_at 8-Sep (SEPT8), mRNA septin 11 (SEPTI l),

NM_018243_at 11-Sep 11-Sep mRNA NM_018243 adenosine deaminase

NM 000022 at ADA ADA (ADA), mRNA NM_000022 adrenomedullin (ADM),

NMJ)Ol 124_at ADM ADM mRNA NMJ)Ol 124 adhesion molecule with

Ig-like domain 2

NM_181847_at AMIGO2 AMIGO2 (AMIGO2), mRNA NMJ 81847 anthrax toxin receptor 2

NM_058172_at ANTXR2 ANTXR2 (ANTXR2), mRNA NM_058172

Rho GTPase activating

ARHGAP protein 1 (ARHGAPl),

NM _004308_at 1 ARHGAPl mRNA NMJ)04308

Rho GTPase activating

ARHGAP protein 22 (ARHGAP22),

NM_021226_at 22 ARHGAP22 mRNA NMJ)21226

Rho GTPase activating

ARHGAP protein 29 (ARHGAP29),

NM_004815_at 29 ARHGAP29 mRNA NM 004815

ATPase, Class I, type 8B, member 2 (ATP8B2), transcript variant 1, NM 00100585

NM_020452_at ATP8B2 ATP8B2 mRNA 5

ATPase, Class II, type 9A

NM_006045_at ATP9A ATP9A (ATP9A), mRNA NMJ)06045 brain abundant, membrane attached signal protein 1 (BASPl),

NM_006317_at BASPl BASPl mRNA NMJ)06317 basonuclin 1 (BNCl),

NM_001717_at BNCl BNCl mRNA NMJ)01717 basonuclin 2 (BNC2),

NM_017637_at BNC2 BNC2 mRNA NM O 17637 chromosome 20 open reading frame 42

NM_017671_at C20orf42 C20orf42 (C20orf42), mRNA NM O 17671 chromosome 8 open reading frame 13

NM_053279_at C8orfl3 C8orfl3 (C8orfl3), mRNA NMJ)53279 calpain 1, (mu/I) large

NM_005186_at CAPNl CAPNl subunit (CAPNl), mRNA NMJ)05186

Cas-Br-M (murine) ecotropic retroviral transforming sequence b

NM_170662_at CBLB CBLB (CBLB), mRNA NMJ 70662

CD55 molecule, decay accelerating factor for complement (Cromer blood group) (CD55),

NM_000574_at CD55 CD55 mRNA NMJ)00574 cadherin 11, type 2, OB- cadherin (osteoblast)

NMJ)01797_at CDHI l CDHI l (CDHI l), mRNA NMJ)01797

NM 001257 at CDHl 3 CDHl 3 cadherin 13. H-cadherin NM 001257 (heart) (CDH13), mRNA carbohydrate (N- acetylglucosamine-6-O) sulfotransferase 2

NM_004267_at CHST2 CHST2 (CHST2), mRNA NMJ)04267 collagen, type VI, alpha 1

NM_001848_at COL6A1 COL6A1 (COL6A1), mRNA NMJ)01848 collagen, type VI, alpha 2

(COL6A2), transcript

NMJ)01849_at COL6A2 COL6A2 variant 2C2, mRNA NMJ)01849 collagen, type VI, alpha 3

(COL6A3), transcript

NM_004369_at COL6A3 COL6A3 variant l, mRNA NMJW4369 collagen, type VI, alpha 3

(COL6A3), transcript

NM_057164_at COL6A3 COL6A3 variant 2, mRNA NMJ)04369 collagen, type VI, alpha 3

(COL6A3), transcript

NM_057165_at COL6A3 COL6A3 variant 3, mRNA NMJW4369 collagen, type VI, alpha 3

(COL6A3), transcript

NM_057166_at COL6A3 COL6A3 variant 4, mRNA NMJ)04369 collagen, type VI, alpha 3

(COL6A3), transcript

NM_057167_at COL6A3 COL6A3 variant 5, mRNA NMJ)04369 chondroitin sulfate synthase 3 (CSS3),

NM_175856_at CSS3 mRNA NMJ75856 dapper, antagonist of beta-catenin, homolog 1

(Xenopus laevis)

NM_016651_at DACTl DACTl (DACTl), mRNA NMJ) 16651 aspartyl aminopeptidase

NM_012100_at DNPEP DNPEP (DNPEP), mRNA NMJH2100 downregulated in ovarian cancer 1 (DOCl), transcript variant 3, NM 00104245

NMJ)01042459_at DOCl FILIPlL mRNA 9 downregulated in ovarian cancer 1 (DOCl), transcript variant 2, NM 00104245

NM_014890_at DOCl FILIPlL mRNA 9 downregulated in ovarian cancer 1 (DOCl), transcript variant 1 , NM 00104245

NM_182909_at DOCl FILIPlL mRNA 9 dedicator of cytokinesis

NM_014689_at DOCKlO DOCKlO 10 (DOCKlO), mRNA NMJH4689 dihydropyrimidinase-like

NM 001387 at DPYSL3 DPYSL3 3 (DPYSL3), mRNA NM 001387 engrailed homolog 1

NMJ)01426_at ENl ENl (ENl), mRNA NMJ)01426

ERBB receptor feedback inhibitor 1 (ERRFIl),

NM_018948_at ERRFIl ERRFIl mRNA NM O 18948 exostoses (multiple) 1

NM_000127_at EXTl EXTl (EXTl), mRNA NM OOO 127 fibrillin 2 (congenital contractural arachnodactyly) (FBN2),

NM_001999_at FBN2 FBN2 mRNA NMJ)01999

FYVE, RhoGEF and PH domain containing 6

NM_018351_at FGD6 FGD6 (FGD6), mRNA NMJ) 18351 forkhead box Dl

NM_004472_at FOXDl FOXDl (FOXDl), mRNA NM 004472 forkhead box Kl NM 00103716

NMJ)Ol 037165_at FOXKl FOXKl (FOXKl), mRNA 5 forkhead box Pl

(FOXPl), transcript NM 00101250

NM_032682_at FOXPl FOXPl variant 1, mRNA 5 frizzled homolog 7

(Drosophila) (FZD7),

NM_003507_at FZD7 FZD7 mRNA NM_003507

GATA binding protein 6

NM_005257_at GATA6 GATA6 (GATA6), mRNA NM_005257 glutamine-fructose-6- phosphate transaminase 2

NM_005110_at GFPT2 GFPT2 (GFPT2), mRNA NM_005110 hyaluronan synthase 2

NMJ)05328_at HAS2 HAS2 (HAS2), mRNA NM_005328 histamine receptor Hl

NM_000861_at HRHl HRHl (HRHl), mRNA NM_000861 heparan sulfate

(glucosamine) 3-0-

HS3ST3A sulfotransferase 3Al

NM_006042_at 1 HS3ST3A1 (HS3ST3A1), mRNA NM_006042 insulin-like growth factor binding protein 3

(IGFBP3), transcript

NM_000598_at IGFBP3 IGFBP3 variant 2, mRNA NMJJ00598 insulin-like growth factor binding protein 3

(IGFBP3), transcript

NM_001013398_at IGFBP3 IGFBP3 variant l, mRNA NM_000598 iroquois homeobox

NM_033267_at IRX2 IRX2 protein 2 (IRX2), mRNA NM_033267 iroquois homeobox

NM_024336_at IRX3 IRX3 protein 3 (IRX3), mRNA NM 024336 integrin, beta 5 (ITGB5),

NM 002213 at ITGB5 ITGB5 mRNA NM 002213 potassium large conductance calcium- activated channel, subfamily M, alpha member 1 (KCNMAl), transcript variant 1 , NM_00101479

NM 001014797 at KCNMAl KCNMAl mRNA 7 potassium large conductance calcium- activated channel, subfamily M, alpha member 1 (KCNMAl), transcript variant 2, NM OO 101479

NM_002247_at KCNMAl KCNMAl mRNA 7

PREDICTED: KIAA 1462, transcript

KIAA 146 variant 1 (KIAA 1462), XM_166132_at 2 KIAA 1462 mRNA XMJ66132

PREDICTED: KLAA 1462, transcript

KIAA 146 variant 2 (KIAA 1462),

XM_929312_at 2 KIAA1462 mRNA XMJ66132

PREDICTED: KIAA1462, transcript XM_933582_at KIAA1462 variant 3 (KIAA 1462), mRNA PREDICTED: KIAA1462, transcript XM_933586_at KIAA 1462 variant 4 (KIAA 1462), mRNA PREDICTED: KIAA 1462, transcript

KIAA 146 variant 5 (KIAA 1462),

XM_940811_at 2 KIAA 1462 mRNA XMJ66132

PREDICTED: KIAA 1462, transcript XM_944813_at KIAA1462 variant 6 (KIAA 1462), mRNA PREDICTED: KIAA 1462, transcript XM 944815 at KIAA 1462 variant 7 (KIAA 1462), mRNA PREDICTED: KIAA 1462, transcript

KIAA 146 variant 8 (KIAA 1462),

XM_944819_at 2 KIAA 1462 mRNA XM 166132

KIAA191 KIAA1913 (KIAA1913),

NM_052913_at 3 KIAA1913 mRNA NM_052913 leprecan-like 1

NM 018192 at LEPRELl LEPRELl (LEPRELl), mRNA NMJ)18192 hypothetical protein

LOC2863 LOC286334 NM OO 104006

NM_ _001040063_at 34 ZNF618 (LOC286334), mRNA 3 hypothetical protein

LOC3400 LOC340061

NM_ _198282_at 61 TMEM 173 (LOC340061), mRNA NM_198282

PREDICTED: similar to

LOC6456 WDNMl homolog

XM 928653 at 38 (LOC645638), mRNA XM_928653 PREDICTED: similar to

LOC6506 WDNMl homolog

XM_939715_at 26 (LOC650626), mRNA XM 939715

PREDICTED: similar to ATPase,

LOC6535 Class II, type 9A (LOC653502),

XM_927759_at 02 mRNA

PREDICTED: similar to ATPase,

LOC6540 Class II, type 9A (LOC654090),

XM_939088_at 90 mRNA lysyl oxidase-like 1

NM_005576_at LOXLl LOXLl (LOXLl), mRNA NM_005576

NM_004811_at LPXN LPXN leupaxin (LPXN), mRNA NM_004811 low density lipoprotein receptor-related protein

NM_032832_at LRPI l LRPI l 11 (LRPI l), mRNA NM_032832 low density lipoprotein receptor-related protein 4

NM_002334_at LRP4 LRP4 (LRP4), mRNA NM_002334 mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALTl), transcript variant 1,

NM_006785_at MALTl MALTl mRNA NM_006785 mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALTl), transcript variant 2,

NM_173844_at MALTl MALTl mRNA NM 006785

Meisl, myeloid ecotropic viral integration site 1 homolog (mouse)

NM_002398_at MEISl MEISl (MEISl), mRNA NM_002398

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_002399_at MEIS2 MEIS2 variant f, mRNA NM_002399

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_020149_at MEIS2 MEIS2 variant e, mRNA NM 002399

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_170674_at MEIS2 MEIS2 variant b, mRNA NM 002399

Meisl, myeloid ecotropic

NM 170675 at MEIS2 MEIS2 viral integration site 1 NM 002399 homolog 2 (mouse)

(MEIS2), transcript variant c, mRNA

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM 170676 at MEIS2 MEIS2 variant d, mRNA NM_002399

Meis 1 , myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_170677_at MEIS2 MEIS2 variant a, mRNA NM 002399

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_172315_at MEIS2 MEIS2 variant g, mRNA NM 002399

Meisl, myeloid ecotropic viral integration site 1 homolog 2 (mouse)

(MEIS2), transcript

NM_172316_at MEIS2 MEIS2 variant h, mRNA NM_002399 myelin protein zero-like 1

(MPZLl), transcript

NM_003953_at MPZLl MPZLl variant 1, mRNA NM 003953 myelin protein zero-like 1

(MPZLl), transcript

NM_024569_at MPZLl MPZLl variant 2, mRNA NM_003953 myosin VI (MY06),

NM_004999_at MYO6 MYO6 mRNA NM_004999 nucleolar protein with

MIF4G domain 1

NM_138400_at NOMl NOMl (NOMl), mRNA NMJ38400

5 '-nucleotidase domain containing 1 (NT5DC1),

NM_152729_at NT5DC1 NT5DC1 mRNA NM l 52729

PREDICTED: odz, odd Oz/ten-m homolog 4 (Drosophila) (ODZ4),

XM_166254_at ODZ4 mRNA

PREDICTED: odz, odd

Oz/ten-m homolog 4

(Drosophila) (ODZ4), XM OO 113114

XM_940463_at ODZ4 ODZ4 mRNA 4

3'-phosphoadenosine 5'- phosphosulfate synthase 2

(PAPSS2), transcript NM_00101588

NMJ)OlOl 5880_at PAPSS2 PAPSS2 variant 2, mRNA 0

3'-phosphoadenosine 5'- phosphosulfate synthase 2 NM_00101588

NM 004670 at PAPSS2 PAPSS2 (PAPSS2), transcript 0 variant 1 , mRN A pre-B-cell leukemia transcription factor 1

NM_002585_at PBXl PBXl (PBXl), mRNA NM 002585 protocadherin 18 NM_019035_at PCDH 18 PCDH 18 (PCDH 18), mRNA NM_019035 procollagen C- endopeptidase enhancer

NM_002593_at PCOLCE PCOLCE (PCOLCE), mRNA NM_002593 procollagen C-

PCOLCE endopeptidase enhancer 2 NM_013363_at 2 PCOLCE2 (PCOLCE2), mRNA NM_013363 phosphodiesterase 5A, cGMP-specific (PDE5A), transcript variant 2,

NM_033430_at PDE5A PDE5A mRNA NMJ)01083 platelet-derived growth factor receptor, alpha polypeptide (PDGFRA),

NM_006206_at PDGFRA PDGFRA mRNA NM_006206 protein inhibitor of activated STAT, 3 NM_006099_at PIAS3 PIAS3 (PIAS3), mRNA NM_006099 paired-like homeodomain transcription factor 2 (PITX2), transcript

NM_000325_at PITX2 PITX2 variant 3, mRNA NM 000325 paired-like homeodomain transcription factor 2 (PITX2), transcript NM_153426_at PITX2 PITX2 variant 2, mRNA NM_000325 paired-like homeodomain transcription factor 2 (PITX2), transcript

NM_153427_at PITX2 PITX2 variant 1 , mRNA NM_000325 plasminogen activator, urokinase (PLAU), NM_002658_at PLAU PLAU mRNA NM_002658 paired related homeobox 1 (PRRXl), transcript NM_006902_at PRRXl PRRXl variant pmx-la, mRNA NM_006902 paired related homeobox 1 (PRRXl), transcript NM_022716_at PRRXl PRRXl variant pmx-lb, mRNA NM 006902 proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2),

NM_024430_at PSTPIP2 PSTPIP2 mRNA NM 024430 prostaglandin E synthase NM 004878 at PTGES PTGES (PTGES), mRNA NM 004878 prostaglandin F2 receptor negative regulator

NM 020440 at PTGFRN PTGFRN (PTGFRN), mRNA NM 020440 prostaglandin- endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)

NM 000963 at PTGS2 PTGS2 (PTGS2), mRNA NM 000963 protein tyrosine phosphatase, receptor

NM 002841 at PTPRG PTPRG type, G (PTPRG), mRNA NM 002841 protein tyrosine phosphatase, receptor type, M (PTPRM),

NM 002845 at PTPRM PTPRM mRNA NM 002845

RNA binding motif protein 9 (RBM9), transcript variant 1, NMJ)0103169

NM 001031695 at RBM9 RBM9 mRNA 5

RNA binding motif protein 9 (RBM9), transcript variant 2, NM 00103169

NM 014309 at RBM9 RBM9 mRNA 5 recombining binding protein suppressor of hairless (Drosophila) (RBPSUH), transcript

NM 005349 at RBPSUH RBPSUH variant 1 , mRNA NM 005349 recombining binding protein suppressor of hairless (Drosophila) (RBPSUH), transcript

NM 015874 at RBPSUH RBPSUH variant 2, mRNA NM 005349 recombining binding protein suppressor of hairless (Drosophila) (RBPSUH), transcript

NM 203283 at RBPSUH RBPSUH variant 3, mRNA NM 005349 recombining binding protein suppressor of hairless (Drosophila) (RBPSUH), transcript

NM 203284 at RBPSUH RBPSUH variant 4, mRNA NM 005349 regulator of G-protein signalling 3 (RGS3), transcript variant 2,

NM 021106 at RGS3 RGS3 mRNA NM 017790 regulator of G-protein signalling 3 (RGS3),

NM 130795 at RGS3 RGS3 transcript variant 1, NM 017790 mRNA regulator of G-protein signalling 3 (RGS3), transcript variant 4,

NM_134427_at RGS3 RGS3 mRNA NM_017790 regulator of G-protein signalling 3 (RGS3), transcript variant 6,

NM_144488_at RGS3 RGS3 mRNA NM_017790 regulator of G-protein signalling 3 (RGS3), transcript variant 5,

NM_144489_at RGS3 RGS3 mRNA NMJ) 17790

Ras-induced senescence 1

NM_015444_at RISl TMEMl 58 (RISl), mRNA NM O 15444 roundabout, axon guidance receptor, homolog 1 (Drosophila)

(ROBOl), transcript

NM_002941_at ROBOl ROBOl variant 1, mRNA NM_002941 roundabout, axon guidance receptor, homolog 1 (Drosophila)

(ROBOl), transcript

NM_133631_at ROBOl ROBOl variant 2, mRNA NM_002941

RYK receptor-like tyrosine kinase (RYK), transcript variant 1, NM 00100586

NM_001005861_at RYK RYK mRNA 1

RYK receptor-like tyrosine kinase (RYK), transcript variant 2, NM 00100586

NM 002958 at RYK RYK mRNA 1 serpin peptidase inhibitor, clade B (ovalbumin),

SERPINfB member 2 (SERPINB2),

NM 002575 at 2 SERPINB2 mRNA NM_002575 sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) (SGCD), transcript variant 1,

NM_000337_at SGCD SGCD mRNA NM_000337

NM_020859_at SHRM SHROOM3 shroom (SHRM), mRNA NM_020859 solute carrier family 16

(monocarboxylic acid transporters), member 7

NM_004731_at SLC16A7 SLC16A7 (SLC 16A7), mRNA NM_004731 solute carrier family 25

SLC25A1 (mitochondrial carrier,

NM 003705 at 2 SLC25A12 Aralar), member 12 NM 003705 (SLC25A12), mRNA solute carrier family 38, member 1 (SLC38A1),

NM 030674 at SLC38A1 SLC38A1 mRNA NM 030674

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 3 (SMARCD3),

SMARCD transcript variant 3, NMJ)0100380 NM 001003801 at 3 SMARCD3 mRNA 1

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 3 (SMARCD3),

SMARCD transcript variant 1, NM_00100380 NM 001003802 at 3 SMARCD3 mRNA 1

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 3 (SMARCD3),

SMARCD transcript variant 2, NMJ)0100380 NM 003078 at 3 SMARCD3 mRNA 1

SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal)

NM_000346_at SOX9 SOX9 (SOX9), mRNA NM 000346 STAM binding protein-

STAMBP like 1 (STAMBPLl), NM 020799 at Ll STAMBPLl mRNA NM 020799 six transmembrane epithelial antigen of the prostate 1 (STEAPl),

NM 012449 at STEAPl STEAPl mRNA NM 012449 six transmembrane epithelial antigen of the prostate 2 (STEAP2), transcript variant 2, NM OO 104066

NM 001040665 at STEAP2 STEAP2 mRNA 5 six transmembrane epithelial antigen of the prostate 2 (STEAP2), transcript variant 3, NMJ)0104066

NM 001040666 at STEAP2 STEAP2 mRNA 5 six transmembrane epithelial antigen of the prostate 2 (STEAP2), NMJ)0104066

NM 152999 at STEAP2 STEAP2 transcript variant 1, 5 mRNA

T-box 3 (ulnar mammary syndrome) (TBX3), transcript variant 1 ,

NM 005996 at TBX3 TBX3 mRNA NM 005996

T-box 3 (ulnar mammary syndrome) (TBX3), transcript variant 2,

NM 016569 at TBX3 TBX3 mRNA NM 005996 tissue factor pathway inhibitor (lipoprotein- associated coagulation inhibitor) (TFPI), transcript variant 2, NM 00103228

NM 001032281 at TFPI TFPI mRNA 1 tissue factor pathway inhibitor (lipoprotein- associated coagulation inhibitor) (TFPI), transcript variant 1, NM OO 103228

NM_006287_at TFPI TFPI mRNA 1 thrombospondin, type I, domain containing 4

NM_024817_at THSD4 THSD4 (THSD4), mRNA NM_024817

Thy-1 cell surface antigen

NM_006288_at THYl THYl (THYl), mRNA NM_006288

TCDD-inducible poly(ADP-ribose) polymerase (TIPARP),

NM_015508_at TIPARP TIPARP mRNA NM O 15508 transducin-like enhancer of split 4 (E(spl) homolog, Drosophila)

NM 007005 at TLE4 TLE4 (TLE4), mRNA NM_007005 tumor necrosis factor receptor superfamily,

TNFRSF2 member 21 (TNFRSF21),

NM_014452_at 1 TNFRSF21 mRNA NM_014452 trophoblast glycoprotein NM_006670_at TPBG TPBG (TPBG), mRNA NM_006670

TRAF3 interacting protein 2 (TRAF3IP2),

TRAF3IP transcript variant 1,

NM 147200 at 2 TRAF3IP2 mRNA NMJ47200

TRAF3 interacting protein 2 (TRAF3IP2),

TRAF3IP transcript variant 2, NM 147686 at 2 TRAF3IP2 mRNA NM 147200 twist homolog 1

(acrocephalosyndactyly 3;

NM 000474 at TWISTl TWISTl Saethre-Chotzen NM 000474 syndrome) (Drosophila)

(TWISTl), mRNA twist homolog 2

(Drosophila) (TWIST2),

NM_057179_at TWIST2 TWIST2 mRNA NM_057179 ubiquitin B (UBB),

NM_018955_at UBB UBB mRNA NM_018955 unc-5 homolog B (C. elegans) (UNC5B),

NM_170744_at UNC5B UNC5B mRNA NM_170744 ubiquitin specific peptidase 3 (USP3),

NM_006537_at USP3 USP3 mRNA NM_006537 wingless-type MMTV integration site family, member 5A (WNT5A),

NM_003392_at WNT5A WNT5A mRNA NM 003392 wingless-type MMTV integration site family, member 5B (WNT5B), transcript variant 2,

NM_030775_at WNT5B WNT5B mRNA NM_030775 wingless-type MMTV integration site family, member 5B (WNT5B), transcript variant 1,

NM_032642_at WNT5B WNT5B mRNA NM_030775 sterile alpha motif and leucine zipper containing kinase AZK (ZAK), transcript variant 1 ,

NM_016653_at ZAK mRNA NM 016653 sterile alpha motif and leucine zipper containing kinase AZK (ZAK), transcript variant 2,

NM_133646_at ZAK mRNA NM 016653 zinc finger protein 618

NM 133374 at ZNF618 ZNF618 (ZNF618), mRNA NM 133374

[00190] Kits [00191] The invention further includes kits combining, in different combinations, high-density oligonucleotide arrays, reagents for use with the arrays, signal detection and array-processing instruments, gene expression databases and analysis and database management software described above. The kits may be used, for example, to predict or model the toxic response of a test compound, to monitor the progression of nerve disease states, to identify genes that show promise as new drug targets and to screen known and newly designed drugs as discussed above.

[00192] The databases packaged with the kits are a compilation of expression patterns from human or laboratory animal genes and gene fragments (corresponding to the genes of Tables 1-3). Data is collected from a repository of both normal and diseased animal tissues and provides reproducible, quantitative results, i.e., the degree to which a gene is up- regulated or down-regulated under a given condition.

[00193] The kits may used in the pharmaceutical industry, where the need for early drug testing is strong due to the high costs associated with drug development, but where bioinformatics, in particular gene expression informatics, is still lacking. These kits will reduce the costs, time and risks associated with traditional new drug screening using cell cultures and laboratory animals. The results of large-scale drug screening of pre- grouped patient populations, pharmacogenomics testing, can also be applied to select drugs with greater efficacy and fewer side-effects. The kits may also be used by smaller biotechnology companies and research institutes who do not have the facilities for performing such large-scale testing themselves. [00194] Databases and software designed for use with microarrays are discussed in

Balaban et al., U.S. Pat. No. Nos. 6,229,911, a computer-implemented method for managing information, stored as indexed tables, collected from small or large numbers of microarrays, and U.S. Pat. No. 6,185,561, a computer-based method with data mining capability for collecting gene expression level data, adding additional attributes and reformatting the data to produce answers to various queries. Chee et al., U.S. Pat. No. 5,974,164, disclose a software-based method for identifying mutations in a nucleic acid sequence based on differences in probe fluorescence intensities between wild type and mutant sequences that hybridize to reference sequences.

[00195] Diagnostic Uses for the Neurofibromatosis and/or MPNST or Sarcoma

Markers

[00196] As described above, the genes and gene expression information provided in Tables 1-3 may be used as diagnostic markers for the prediction or identification of the malignant state of the nerve tissue. For instance, a nerve tissue sample or other sample from a patient may be assayed by any of the methods described above, and the expression levels from a gene or genes from the Tables, in particular the genes in Table 1 , may be compared to the expression levels found in normal nerve tissue, tissue from or neurofibromatosis and/or MPNST tissue. Expression profiles generated from the tissue or other sample that substantially resemble an expression profile from normal or diseased nerve tissue may be used, for instance, to aid in disease diagnosis. Comparison of the expression data, as well as available sequence or other information may be done by researcher or diagnostician or may be done with the aid of a computer and databases as described above. [00197] Use of the Neurofibromatosis and/or MPNST or Sarcoma Markers for

Monitoring Disease Progression

[00198] As described above, the genes and gene expression information provided in Tables 1-3 may also be used as markers for the monitoring of disease progression, for instance, the development of neurofibromatosis and/or MPNST. For instance, a nerve tissue sample or other sample from a patient may be assayed by any of the methods described above, and the expression levels in the sample from a gene or genes from Tables 1 -3 may be compared to the expression levels found in normal nerve tissue, tissue from malignant neurofibromatosis and/or MPNST or neurofibromatosis and/or MPNST tissue. Comparison of the expression data, as well as available sequence or other information may be done by researcher or diagnostician or may be done with the aid of a computer and databases as described above.

[00199] Use of the Neurofibromatosis and/or MPNST Markers for Drug Screening

[00200] According to the present invention, the genes identified in Tables 1 -3 may be used as markers to evaluate the effects of a candidate drug or agent on a cell, particularly a cell undergoing malignant transformation, for instance, a neurofibromatosis and/or MPNST cell or tissue sample. A candidate drug or agent can be screened for the ability to simulate the transcription or expression of a given marker or markers (drug targets) or to down- regulate or counteract the transcription or expression of a marker or markers. According to the present invention, one can also compare the specificity of drugs' effects by looking at the number of markers which the drugs have and comparing them. More specific drugs will have fewer transcriptional targets. Similar sets of markers identified for two drugs indicate a similarity of effects. [00201] Assays to monitor the expression of a marker or markers as defined in

Tables 1-3 may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down-regulating expression of the nucleic acid in a cell.

[00202] In one assay format, gene chips containing probes to at least two genes from Tables 1-3 may be used to directly monitor or detect changes in gene expression in the treated or exposed cell as described in more detail above. In another format, cell lines that contain reporter gene fusions between the open reading frame and/or the 3' or 5' regulatory regions of a gene in Tables 1-3 and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al., (1990) Anal. Biochem. 188, 245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of the nucleic acid.

[00203] Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a gene identified in Tables 1-3. For instance, as described above, mRNA expression may be monitored directly by hybridization of probes to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al., (1989) Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press). [00204] In another assay format, cells or cell lines are first identified which express the gene products of the invention physiologically. Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades. Such cell lines may be, but are not required to be, derived from nerve tissue. Further, such cells or cell lines may be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 '-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag. Such a process is well known in the art (see Sambrook et al., (1989) Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press).

[00205] Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions; for example, the agent comprises a pharmaceutically acceptable excipient and is contacted with cells comprised in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and serum incubated at 37⁰C. Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent-contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.

[00206] Another embodiment of the present invention provides methods for identifying agents that modulate the levels, concentration or at least one activity of a protein(s) encoded by the genes in Tables 1-3. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

[00207] In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an unexposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe, such as a specific antibody.

[00208] Agents that are assayed in the above methods can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

[00209] As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.

[00210] For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.

[00211] The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNA encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic" as used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant, (1995) in Molecular Biology and Biotechnology Meyers (editor) VCH Publishers). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

[00212] Test Compounds and Extracts

[00213] A recent trend in medicinal chemistry includes the production of mixtures of compounds, referred to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. 1992. J. Am. Chem. Soc. 114:10987; De Witt et al. 1993. Proc. Natl. Acad. Sci. USA 90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993. Science. 261 :1303), and hydantoins (De Witt et al. supra). Rebek et al. have described an approach for the synthesis of molecular libraries of small organic molecules with a diversity of 104-105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).

[00214] The compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one-bead one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. Anticancer Drug Des. 1997. 12:145).

[00215] In one embodiment, the test compound is a peptide or peptidomimetic. In another, preferred embodiment, the compounds are small, organic non- peptidic compounds.

[00216] Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb et al. 1994. Proc. Natl. Acad. Sci. USA 91 :11422; Horwell et al. 1996 Immunopharmacology 33:68; and in Gallop et al. 1994. J. Med. Chem. 37:1233. In addition, libraries such as those described in the commonly owned applications U.S. Ser. No. 08/864,241, U.S. Ser. No. 08/864,240 and U.S. Ser. No. 08/835,623 can be used to provide compounds for testing in the present invention. The content of each of these applications is expressly incorporated herein by this reference.

[00217] Libraries of compounds may be presented in solution (e.g., Houghten

(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. MoI. Biol. 222:301-310); (Ladner supra.).

[00218] In certain embodiments, the test compounds are exogenously added to the modified cells expressing a recombinant receptor and compounds that modulate signal transduction via the receptor are selected. In other embodiments, the modified cells express the compounds to be tested. For example, a culture of the subject modified cells can be further modified to collectively express a peptide library as described in more detail in PCT Publication WO 94/23025 the contents of which is expressly incorporated herein by this reference.

[00219] Other types of peptide libraries may also be expressed, see, for example,

U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publication WO94/ 02502). In still another embodiment, the combinatorial polypeptides are produced from a cDNA library.

[00220] Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries. [00221] In general, compounds are identified from large libraries of both natural product and synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

[00222] In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their anti-pathogenic activity should be employed whenever possible.

[00223] When a crude extract is found to have activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having the desired activity. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art.

[00224] Pharmaceutical Therapeutics

[00225] The invention provides a simple means for identifying compounds

(including peptides, small molecule inhibitors, and mimetics) capable of inhibiting the pathogenicity or virulence of a pathogen. Accordingly, chemical entities discovered to have medicinal or agricultural value using the methods described herein are useful as either drugs, plant protectants, or as information for structural modification of existing anti-pathogenic compounds, e.g., by rational drug design. Such methods are useful for screening compounds having an effect on a variety of pathogens including, but not limited to, bacteria, viruses, fungi, annelids, nematodes, Platyhelminthes, and protozoans. Examples of pathogenic fungi include, without limitation, Candida albicans, Aspergillus sp, Mucor sp, Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp. Cryptococcis neoformans, Pneumocystis carinii, and Trichophyton sp. [00226] For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Treatment may be accomplished directly, e.g., by treating the animal with antagonists that disrupt, suppress, attenuate, or neutralize the biological events associated with a pathogenicity polypeptide. Preferable routes of administration include, for example, inhalation or subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of an anti-pathogenic agent in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the anti -pathogenic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease and extensiveness of the disease. Generally, amounts will be in the range of those used for other agents used in the treatment of other microbial diseases, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that inhibits microbial proliferation. For example, for systemic administration a compound is administered typically in the range of 0.1 ng-10 g/kg body weight.

[00227] In addition, the antipathogenic agent may be added to materials used to make catheters, including but not limited to intravenous, urinary, intraperitoneal, ventricular, spinal and surgical drainage catheters, in order to prevent colonization and systemic seeding by potential pathogens. Similarly, the antipathogenic agent may be added to the materials that constitute various surgical prostheses and to dentures to prevent colonization by pathogens and thereby prevent more serious invasive infection or systemic seeding by pathogens.

[00228] Other Embodiments

[00229] In general, the invention includes any nucleic acid sequence which may be isolated as described herein or which is readily isolated by homology screening or PCR amplification using the nucleic acid sequences of the invention. Also included in the invention are polypeptides which are modified in ways which do not abolish their pathogenic activity (assayed, for example as described herein). Such changes may include certain mutations, deletions, insertions, or post-translational modifications, or may involve the inclusion of any of the polypeptides of the invention as one component of a larger fusion protein.

[00230] Thus, in other embodiments, the invention includes any protein which is substantially identical to a polypeptide of the invention. Such homologs include other substantially pure naturally-occurring polypeptides as well as allelic variants; natural mutants; induced mutants; proteins encoded by DNA that hybridizes to any one of the nucleic acid sequences of the invention under high stringency conditions or, less preferably, under low stringency conditions (e.g., washing at 2x SSC at 40 C. with a probe length of at least 40 nucleotides); and proteins specifically bound by antisera of the invention.

[00231] The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from the naturally- occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally- occurring amino acid sequence of the invention. The length of sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^{" 100} indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta or gamma amino acids. In addition to full-length polypeptides, the invention also includes fragments of any one of the polypeptides of the invention. As used herein, the term "fragment," means at least five, preferably at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[00233] Chemical Screen

[00234] Compounds. A subset of chemically diverse compounds assembled to broadly cover chemical space within the areas considered drug-like, filtered to avoid functional groups with known toxicity or stability issues can be used in accordance with the described methods.

[00235] For example, compounds that are: 1) unlikely to be toxic or chemically reactive, 2) soluble, and 3) similar in molecular size and structure to current marketed drugs might be selected for screening using the described compositions and methods. The most potent (<200) of these compounds may be selected. A broader compound database may be sifted for the most similar compounds (nearest neighbors) as defined by a number of computational terms. Screening of these compounds (generally about 1000) should lead to more potent hits. Several iterations of this cycle would be expected to allow identification of the most potent compounds in the broad library through screening of fewer compounds.

[00236] The lack of structural or class information on targets associated with NF 1 loss leaves broad-based screening as the optimal approach. This approach to screening allows identification of a collection of relatively drug-like hits for further study.

[00237] Pharmaceutical Compositions

[00238] After identifying certain test compounds in the subject assay as potential therapeutic agents, e.g., as potential therapeutic agents for the treatment of an NFl -related disorder or condition, the practitioner of the subject assay will continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo. Whether for subsequent in vivo testing, or for administration to an animal as an approved drug, agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human. The compounds selected in the subject assay, or a pharmaceutically acceptable salt thereof, may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the compound, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on the above, such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the compound in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. In preferred embodiment, the compound can be disposed in a sterile preparation for topical and/or systemic administration. In the case of freeze-dried preparations, supporting excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH. Similar solutions may also be used for the pharmaceutical compositions of compounds in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).

[00240] Another aspect of this invention is compositions that comprise a safe and effective amount of a subject compound, or a pharmaceutically-acceptable salt thereof, and a pharmaceutically-acceptable carrier. As used herein, "safe and effective amount" means an amount of the subject compound sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. A safe and effective amount of the subject compound will vary with the age and physical condition of the patient being treated, severity of the condition, duration of the treatment, the nature of concurrent therapy, the particular pharmaceutically-acceptable carrier utilized, and like factors within the knowledge and expertise of the attending physician.

[00241] Preparing a dosage form is within the purview of the skilled artisan.

Examples are provided for the skilled artisan, but are non-limiting, and it is contemplated that the skilled artisan can prepare variations of the compositions claimed. - I l l -

[00242] In addition to the subject compound, the compositions of this invention contain a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier," as used herein, means one or more compatible solid or liquid filler diluents or encapsulating substances which are suitable for administration to a mammal. The term "compatible", as used herein, means that the components of the composition are capable of being commingled with the subject compound, and with each other, in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the composition under ordinary use situations. Preferably when liquid dose forms are used, the compounds of the invention are soluble in the components of the composition. Pharmaceutically-acceptable carriers must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the mammal being treated.

[00243] Some examples of substances which can serve as pharmaceutically- acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS®; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the subject compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.

[00244] If the mode of administering the subject compound is perorally, the preferred unit dosage form is therefore tablets, capsules, lozenges, chewable tablets, and the like. Such unit dosage forms comprise a safe and effective amount of the subject compound, which is preferably from about 0.01 mg to about 350 mg, more preferably from about 0.1 mg to about 35 mg, based on a 70 kg person. The pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for peroral administration are well-known in the art. Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention, and can be readily made by a person skilled in the art.

[00245] Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Such liquid oral compositions preferably comprise from about 0.001% to about 5% of the subject compound, more preferably from about 0.01% to about 0.5%. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, AVICEL®RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[00246] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual and buccal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.

[00247] Compositions can also be used to deliver the compound to the site where activity is desired: intranasal doses for nasal decongestion, inhalants for asthma, and eye drops, gels and creams for ocular disorders.

[00248] Preferred compositions of this invention include solutions or emulsions, preferably aqueous solutions or emulsions comprising a safe and effective amount of a subject compound intended for topical intranasal administration. Such compositions preferably comprise from about 0.001% to about 25% of a subject compound, more preferably from about 0.01% to about 10%. Similar compositions are preferred for systemic delivery of subject compounds by the intranasal route. Compositions intended to deliver the compound systemically by intranasal dosing preferably comprise similar amounts of a subject compound as are determined to be safe and effective by peroral or parenteral administration. Such compositions used for intranasal dosing also typically include safe and effective amounts of preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfate and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof, and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions as needed. The compositions may also comprise local anesthetics or other actives. These compositions can be used as sprays, mists, drops, and the like. Other preferred compositions of this invention include aqueous solutions, suspensions, and dry powders comprising a safe and effective amount of a subject compound intended for atomization and inhalation administration. Such compositions preferably comprise from about 0.1% to about 50% of a subject compound, more preferably from about 1% to about 20%; of course, the amount can be altered to fit the circumstance of the patient contemplated and the package. Such compositions are typically contained in a container with attached atomizing means. Such compositions also typically include propellants such as chlorofluorocarbons 12/11 and 12/114, and more environmentally friendly fluorocarbons, or other nontoxic volatiles; solvents such as water, glycerol and ethanol, these include co-solvents as needed to solvate or suspend the active; stabilizers such as ascorbic acid, sodium metabisulfite; preservatives such as cetylpyridinium chloride and benzalkonium chloride: tonicity adjustors such as sodium chloride; buffers; and flavoring agents such as sodium saccharin. Such compositions are useful for treating respiratory disorders, such as asthma and the like.

[00250] Other preferred compositions of this invention include aqueous solutions comprising a safe and effective amount of a subject compound intended for topical intraocular administration. Such compositions preferably comprise from about 0.0001% to about 5% of a subject compound, more preferably from about 0.01% to about 0.5%. Such compositions also typically include one or more of preservatives, such as benzalkonium chloride, thimerosal, phenylmercuric acetate; vehicles, such as poloxamers, modified celluloses, povidone and purified water; tonicity adjustors, such as sodium chloride, mannitol and glycerin; buffers such as acetate, citrate, phosphate and borate; antioxidants such as sodium metabisulfite, butylated hydroxy toluene and acetyl cysteine; acids and bases may be used to adjust the pH of these formulations as needed.

[00251] Other preferred compositions of this invention useful for peroral administration include solids, such as tablets and capsules, and liquids, such as solutions, suspensions and emulsions (preferably in soft gelatin capsules), comprising a safe and effective amount of a subject compound. Such compositions preferably comprise from about 0.01 mg to about 350 mg per dose, more preferably from about 0.1 mg to about 35 mg per dose. Such compositions can be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings, waxes and shellac.

[00252] Any of the compositions of this invention may optionally include other drug actives.

[00253] The invention further provides compositions (e.g., nucleotide sequence probes) and methods for the diagnosis of a pathogenic condition.

[00254] Industrial Applicability

[00255] The gene-expression analysis of neurofibroma, MPNST, and/or other peripheral nerve tumor or sarcoma described herein, has identified specific genes as targets for cancer prevention and therapy. Based on the expression of a subset of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying or detecting MPNST.

[00256] The methods described herein are also useful in the identification of additional molecular targets for prevention, diagnosis and treatment of MPNST. The data reported herein add to a comprehensive understanding of MPNST, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of testicular tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of MPNST.

[00257] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[00258] Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, applications and publications referred to in this application are herein incorporated by reference in their entirety for any purpose.

[00259] Example 1.

[00260] Neurofibroma acquisition

[00261] The diagnosis of NFl was established according to published criteria

(Gutmann et al., 1997). Tumor tissue for solid tumor analysis and paraffin sections was snap frozen at the time of surgery from patients undergoing surgical treatment at Massachusetts General Hospital and was obtained from the Neuro-Oncology Tissue Repository at Massachusetts General Hospital in accordance with IRB-approved protocols. Blocks of corresponding paraffin-embedded tumors were obtained from the MGH- pathology files. Histological review of all specimens was performed by a neuropathologist to ensure that all tumors were classified and graded in a uniform manner according to the current WHO classification. For Schwann cell isolation, we froze tumor in liquid nitrogen (50% DMEM, 40% FBS, 10% DMSO) or directly cultured cells. Live subconfluent purified Schwann cell cultures were shipped to Cincinnati Children's Hospital, incubated in fresh media for 24 hours or until 70 - 80% confluent and flash-frozen for brief storage at -8O⁰C prior to RNA isolation. [00262] Schwann cell isolation

[00263] We generated NHSCs from independent patients as previously described

(Casella et al., 1996). We isolated Schwann cells from dermal neurofibromas as described (Rosenbaum et al., 2000; Serra et al., 2000). Briefly, we mechanically triturated tumor pieces and further digested with enzymes. We resuspended cells in Schwann cell media (SCM: DMEM supplemented with 10% FBS, antibiotics, 0.5mM IBMX, 1OnM beta- heregulin, 0.5 μM forskolin and 2.5μg/ml insulin) and seeded in poly-L- lysine and laminin coated plates. For NFl+/- Schwann cells, forskolin is kept constant (and cells can be maintained for 1 day in N2 serum-free media every 3 - 4 days in SCM). For NFl-/- cells, forskolin is only provided in pulses of 1 day every 3 - 4 days in SCM-no forskolin (cells can also be maintained in N2 serum-free media for 1 day after forskolin pulse). We assessed purity of Schwann cell cultures by immunofluorescent staining with an S-100 antibody and DAPI (Serra et al., 2000). In all cases, > 95% pure Schwann cell cultures were obtained after 3 - 5 passages. When a somatic mutation of a tumor was identified, we assessed purity according to genotype (Supplemental Table 11). In NFl-I- Schwann cell cultures complete LOH was observed. We generated plexiform neurofibroma Schwann cell cultures, 70 - 95% S 100 D -positive, as previously described (Muir et al., 2001; Wallace et al., 2000). These were maintained in media lacking forskolin, to enrich for the NFl -/cells, and were harvested for RNA between passage 4 and 6.

[00264] Mutation analysis in Schwann cells derived from dermal neurofibromas

[00265] We restricted somatic mutation analysis in dNFs to LOH analysis (Serra et al., 2001), comparing DNA from blood and dNF from the same patient. We used several microsatellite markers located within or surrounding the NFl gene for genotyping with ABI 377 and 313Ox Genetic Analyzers. We also analyzed DNA from NFl-I- Schwann cell cultures from tumors exhibiting LOH. Germline mutations of patients with dNFs were identified by the cDNA-SSCP-heteroduplex described elsewhere (Ars et al., 2000).

[00266] Mutation analysis in plexiform neurofibromas and MPNST

[00267] We screened genomic DNA isolated from frozen tissue or cell pellets for

NFl gene mutations by denaturing high-performance liquid chromatography (dHPLC)-based heteroduplex analysis in 13 / 23 solid tumor samples, including dermal neurofibromas (n = 10), plexiform neurofibromas (n = 3) and MPNSTs (n = 1), for which ample high-quality DNA was available, using the WAVE analysis system (Transgenomic; Omaha, NE) (Supplemental Table 11). Primers were designed to reduce their homology to the NFl pseudogenes sequences, and MLPA (Upadhyaya et al., 2006) and sequencing of RTPCR products (Thomson and Wallace, 2002) were employed. We also characterized mutations in 8 / 11 plexiform Schwann cell cultures, using a combination of the above methods. There was no clear association with mutation and tumor type. The MPNST harbored a germline NFl point mutation and a large deletion including the NFl locus as the somatic mutation.

[00268] MPNST cell cultures

[00269] MPNST cell lines ST88-14, STS26T, S520, S462, 88-3, 90-8 and YSTl were grown as described (Miller et al., 2006). We isolated RNA from cells under standard growth conditions for each cell line.

[00270] RNA isolation and microarray hybridization [00271] We isolated total RNA from frozen tissue and cells using the RNeasy kit

(Qiagen) and verified RNA integrity with an Agilent Bioanalyzer 2100 (with typical 28S:18S ratios = 2 ± 0.1). A biotinylated cDNA probe was generated from a single round of DNA displacement synthesis amplification of 20ng total RNA (Ovation Biotin RNA Amplification and Labeling System; NuGen) for hybridization to the whole-genome Affymetrix GeneChip HUl 33 Plus 2.0 using the Affymetrix recommended protocol. We used the Affymetrix Gene Array scanner and GeneChip Operating Software version 1.4 to scan and quantify GeneChips using default settings. We processed total of 86 samples in 9 separate batches, each of which included a universal tissue reference to manage batch-to-batch array variations (see Data analysis strategy for referencing details). Samples were distributed as follows: 9 universal tissue reference samples, 10 normal human Schwann cell (NHSC) samples, 11 dermal neurofϊbroma-derived Schwann cell (dNFSC) samples, 11 plexiform neurofibroma-derived Schwann cell (pNFSC) samples, 13 malignant peripheral nerve sheath tumor cell line (MPNST cell line) samples, 13 dermal neurofibroma (dNF) samples, 13 plexiform neurofibroma (pNF) samples and 6 malignant peripheral nerve sheath tumor (MPNST) samples. We used Affymetrix Microarray Suite 5.0 to generate "CEL" files for each sample that were normalized using the Robust Multichip Analysis (RMA) algorithm as implemented in Bioconductor/R (Irizarry et al., 2003).

[00272] Microarray data quality control and power analysis

[00273] We assessed array images for spatial defect using the GEODEX (Kim et al., 2006) as well as a deleted residuals approach (Persson et al., 2005). Chips with high spatial artifacts or large deviations from outliers were discarded. A total of two experimental chips were discarded: dNF_AS17 and 3_PNF_XT470. In addition, in one instance the normalization control sample failed quality control and the entire batch of samples was rerun.

[00274] Data analysis strategy

[00275] We used a custom GeneChip library file (CDF) based on Refseq target definitions (Hsl33P REFSEQ Version 8) to provide more accurate interpretation of GeneChip data (Dai et al., 2005). To enable the identification of gene expression changes relative to NHSCs, we applied a two-stage referencing strategy: we set the normalized gene expression level value for each transcript in each sample to its ratio relative to the expression of that transcript's measurement in the universal tissue reference; then, we set the normalized gene expression level value for each transcript in each sample to its ratio relative to the median expression of that transcript's measurements across NHSCs. We used the analysis of variance method (ANOVA, P < 0.001) to compare the NHSCs, dNFSCs, pNFSCs and MPNST cell lines. We performed statistical comparisons and data visualization using GeneSpring GX v7.3.1 (Agilent Technologies). We corrected results from the primary analysis for multiple testing effects by applying the Benjamini and Hochberg false discovery rate correction (Benjamini et al., 2001) (FDR < 0.001).

[00276] We assigned transcripts to empirically defined expression patterns using the k-means clustering algorithm. We used Pearson correlation as a similarity measure and genes were iteratively clustered until convergence. After convergence, we tested an additional 5 random clusters to ensure that the optimal clustering solution had been identified. We used k-means clustering to assign genes between principal expression patterns as the k- means clustering algorithm generally finds a clustering solution with a smaller within-cluster sum of distances (meaning a more homogeneous cluster) than hierarchical clustering techniques. For identification of genes sharing common gene expression patterns in cultured cells and solid tumors, we re-clustered each cluster composed of genes that showed increased/decreased expression in dNFSCs, pNFSCs and MPNST cell lines across NHSCs, neurofibromas and MPNSTs. We identified three principle patterns of gene expression and genes were assigned to each using k-means clustering. We pooled sub-clusters displaying a similar pattern of gene expression in neurofibromas and MPNSTs as the original cultured cell cluster with all other similarly expressed sub-clusters, resulting in 1,708 transcripts that were then used to generate a heatmap including the entire sample set, NHSCs, NFSCs, MPNST cell lines, neurofibromas and MPNSTs.

[00277] We identified statistically over-represented Gene Ontologies and both

KEGG and BioCarta pathways (P < 0.05) using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2007 at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (Huang et al., 2007). We used the Gene Association to Anatomic and Clinical Abnormalities (GATACA; web server to select genes based upon association with nerve development, NFl or MPNST.

[00278] Schwann cell development ortholog comparison

[00279] To identify developmental signatures in each of the clusters similarly dysregulated in neurofibroma-related peripheral nerve cell cultures and tumors, we identified genes activated in neural crest cells (E9), several stages of Schwann cell development (immature Schwann cells (E 12 - E 14) and Schwann cell precursors (El 6 - PO) and fully differentiated nerve using two publicly available data sets (Buchstaller et al., 2004; Su et al., 2004) from mouse. We corrected for microarray platform differences and redundancy due to transcript variants and then calculated the overlap between each signature and each cluster. After calculating the expected number of developmental genes for each stage in each cluster, we constructed contingency tables performed a Fisher's exact test for those developmental signatures that occurred greater than expected. Fisher's exact tests were performed with R.

[00280] SOX9 target gene identification

[00281] We used Genome Trafac, a database resource that allows for the genome- wide detection of compositionally similar cw-clusters that occur in gene orthologs between mouse and human, to determine putative SOX9 target genes, identifying SOX9 czs-elements that occurred within an ortholog conserved region ( > 70% sequence similarity based on BlastZ alignment) upstream of the first exon in 2,000 flanking basepairs (Jegga et al., 2007). We then determined the overlap between S OX9 target genes and transcripts similarly dysregulated in neurofibroma-related cultures and tumors to identify 394 unique genes in the entire microarray genome containing potential SOX9 binding sites. We used Fisher's exact test calculate the statistical significance of over-represented SOX9 target genes in the neurofibroma-related culture/tumor gene list.

[00282] Quantitative real time PCR

[00283] We used total RNA as a template to synthesize double-stranded cDNA using an oligo (dT) primer with Superscript III reverse transcriptase (Invitrogen). We conducted duplicate reactions omitting reverse transcriptase to control for genomic DNA contamination. We measured relative levels of RNA by quantitative real time (QRT)-PCR using the ABI 7500 Sequence Detection System default settings. For most genes, we monitored amplification in SYBR Green Master Mix (Applied Biosystems). See Supplemental Table 12 for primer sequences for individual genes. We obtained cycle threshold values where fluorescence intensity was in the geometric phase of amplification and averaged for triplicate reactions. Values for individual genes of interest were normalized to values for beta-actin and used to calculate fold-change in gene expression using ABI software. SOXlO, (ABI TaqMan Probe ID Hs00366918_ml) and 18S (ABI TaqMan Probe ID Hs99999901_sl) were amplified using ABI 2X Universal TaqMan Master Mix (Applied Biosystems). For technical validation of gene expression microarray data, a result of > ± two-fold in the same direction was considered confirmed (Supplemental Table 9).

[00284] Western Analysis

[00285] We lysed cells on ice in 50 mM Tris, pH 7.5, 120 mM NaCl, 1 mM

EDTA, 0.5% NP-40, 0.1 mM sodium vanadate, ImM sodium fluoride, 5 μg per ml leupeptin and 30 μM phenylmethylsulfonyl fluoride. We separated equivalent amounts of protein (100 μg) by electrophoresis on SDS-polyacrylamide gradient gels (7 - 15% or 4 - 20%; ISC BioExpress; Kaysville, UT) and transferred to PVDF membrane (BioRad; Hercules, CA). We probed membranes with anti-SOX9 (Santa Cruz Biotechnology, Santa Cruz, CA; 1 :700), stripped and re-probed with anti-β-actin (Cell Signaling Technology, Inc. #4967) as a loading control. We detected signals using horseradish peroxidase-conjugated secondary antibodies (BioRad; Hercules, CA) in combination with ECL Plus developing system (Amersham Biosciences; Piscataway, NJ) according to manufacturer specifications. For SOX9 quantification, we used ImageJ 1.33u software to obtain values from scanned autoradiographs representing protein levels. We normalized SOX9 levels to β-actin levels for each sample. [00286] Immunohistochemistry

[00287] SOX 9 immunohistochemistry was performed on formalin-fixed, paraffin- embedded sections with a polyclonal anti-SOX9 antibody (Abeam, Cambridge, UK). Antigen retrieval was achieved with microwaving in sodium citrate (pH 6), followed by incubation with the primary antibody overnight at 4°C (1 :75) and visualization with an avidin-biotin complex (Vectastatin Elite ABC kit; Vector Laboratories, Burlingame, CA) and with 3,3'-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA). Histological analysis consisted of scoring SOX9 immunostaining semi-quantitatively for the number of positive cells only, as the staining intensity was similar in all samples (strong). The number of positive cells was scored as < 25% (+1, some), 25% - 75% (+2, many) and > 75% (+3, most). Immunofiuorescent detection of SOX9 in cultured cells was conducted similarly, using a 1 :200 dilution of primary anti- SOX9 antibody followed by a TRITC -conjugated anti-rabbit secondary antibody and DAPI visualization of nuclei.

[00288] Identification of aneuploidy

[00289] We conducted in silico functional aneuploidy identification according to

Callegero et al. (Callegaro et al., 2006). Sex chromosomes were not considered in the analysis. The tabulated data of gains and losses includes Affymetrix probe set ID, the Entrez gene ID, gene symbol, score, smoothed score, chromosome, cytological band (gene map), gene position and q-value (Supplemental Table 7). We used the Affymetrix CDF, as it catalogs multiple probe sets for each gene. The reduction in probe sets (and thus probe coverage) in the Custom CDF reduces the performance of the algorithm to detect chromosomal imbalances. The score column is the original test statistic obtained after performing SAM's two-group comparison (Tusher et al., 2001). A nonparametric regression model was applied to the original t-test statistics to estimate smoothed statistics. If smoothed statistics cross a defined boundary, high or low, this is scored as potential chromosomal gain or loss. Each test statistic is smoothed using regression strategy described in Callegaro et al The non-parametric regression strategy then yields the smoothed score. Filtering was done such that all gains have a fold change > 1 and all losses have fold change < 1. The gains and losses were determined using Storey's FDR (Storey, 2002).

[00290] Lentiviral shRNA infection

[00291] We plated MPNST cells to reach ~ 70 - 90% confluence the next day for infection with lentiviral particles containing vectors with shRNAs targeting SOX9 (Moffat et al., 2006) (Open Biosystems; TRC library) or GFP (Yang et al., 2004) (Addgene) as a non-specific control. Lentiviral shRNA constructs were received as bacterial glycerol stocks for plasmid DNA isolation (Qiagen). The CCHMC Viral Vector Core produced virus using a 4-plasmid packaging system to maximize safety. We incubated lentiviral particles with the MPNST cells (MOI ~ 10) in the presence of polybrene (8 μg/mL; Sigma) for 24 hours followed by selection in puromycin at a concentration (2 μg/mL) that killed uninfected cells plated in parallel within three days. We determined SOX9 expression by QRT- PCR and Western blotting as described above.

[00292] MTS assay

3

[00293] We plated MPNST cells (4 x 10 ) in triplicate and incubated overnight in a 96-well plate for infection with shGFP or shSOX9 lentiviral particles as described above. Cells were maintained in the presence or absence of puromycin (2 μg/μl) to account for infection efficiency. We conducted MTS assays for viable cell number on days 3, 4, 5, 6 and 7 post-infection according to the manufacturer's protocol (Cell Titer 96® Aqueous One Solution Cell Proliferation Assay; Promega).

[00294] TUNEL staining for apoptosis

4

[00295] We plated MPNST cells (2 x 10 ) onto LabTek chamber slides (Nalge-

Nunce International), incubated overnight and fixed in 4% paraformaldehyde for TUNEL detection nuclear DNA fragmentation, indicative of apoptosis, according to the manufacturer's protocol (DeadEnd Fluorometric TUNEL System; Promega; Madison, WI). We plated cells in quadruplicate wells, infected with shSOX9 or shGFP lentiviral particles as described above and assayed for apoptosis after 3 days in the presence or absence of puromycin. One well was used for a negative control, containing no Terminal Deoxynucleotidyl Transferase. We counted cells in five random fields in triplicate wells. FITC positive apoptotic cells are expressed as a percentage of total cells visualized by DAPI.

[00296] Results

[00297] Creation of a comprehensive NFl gene expression dataset

[00298] Neurofibroma tissue samples contain NF 1 +/- and NF 1 -/-Schwann cells, fibroblasts, perineurial cells, endothelial cells and mast cells. To avoid this inherent variability and to describe gene expression changes that correspond to a single cell type, we used purified Schwann cells as the basis for our analysis. To ensure data quality, we minimized non- biological variability in sample batch processing by running samples from each experimental group in each processing batch. To minimize technical variability, we conducted microarray hybridization at a single site, including universal reference RNA in each processing batch as a technical control for batch-to-batch variation. We hybridized samples to whole- genome oligonucleotide microarrays to generate a comprehensive gene expression profile, and took quality control steps at the RNA sample and microarray data levels. Specifically, analysis after each processing batch assessed the power for statistical comparisons (Page et al., 2006). We also used power analysis as a futility analysis in the comparison of dermal and plexiform neurofibromas to determine that a difference between the groups would not be detectable without a far larger sample size (at minimum, 5 times more samples). We redefined microarray probe set annotations to include only valid transcripts (Dai et al., 2005).

[00299] Schwann cell culture transcription profiles distinguish benign from malignant NFl tumors but fail to discriminate neurofibroma subtypes

[00300] To discover gene expression programs that underlie the differences between cultured normal human Schwann cells (NHSCs), dermal and plexiform neurofibroma Schwann cells (dNFSCs and pNFSCs, respectively) and MPNST cell lines, following batch referencing we set gene expression level values for each transcript in each sample to its ratio relative to the median expression of that transcript's measurements across NHSCs. Using parametric statistical analysis (ANOVA, FDR < 0.001), 2,827 transcripts were identified as differentially expressed and subjected to two-way hierarchical tree clustering (Supplemental Table 1). Most MPNST cell lines display a unique transcriptional signature and cluster to the far right of the heatmap, separately from benign neurofibroma-derived Schwann cells. Although we anticipated gene expression signatures that were unique to dNFSCs or pNFSCs, transcripts that passed the ANOVA failed to partition the two neurofibroma subtypes. Direct comparison of dNFSCs and pNFSCs also failed to identify a statistically significant signature. Instead, we observed two classes of neurofibroma Schwann cells (NFSCs), with genes in Class 1 NFSCs (Figure not shown) less up- or down-regulated than genes in Class 2 NFSCs. Class type did not correlate with any known patient parameters or sample handling. Five principal patterns of gene expression were identified and genes were assigned to each using k-means clustering (clusters Cl - C5; Supplemental Table 2). Genes within each cluster were significantly associated with gene function categories using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2007 (see Supplemental Table 3 for biological associations and Supplemental Table 2 for detailed cluster and biological association gene lists).

Schwann cell culture and neurofibromas overex Dressed underexpressed

Significant in observed expected observed expected both comparisons 535 143 na 393 na over-expressed 424 91 113 331 under-expressed 111 m t£M 29 60 82

[00301] Transcripts differentially expressed in NFl tumor cell cultures share gene expression signatures with NFl solid tumors

[00302] In a heatmap of cultured Schwann cells compared to cultured dNFSCs, pNFSCs and MPNST cell lines (figure not shown), unsupervised clustering grouped samples as either normal human Schwann cells (NHSC), mixed benign dermal and plexiform neurofibroma Schwann cells (dNFSC and pNFSC) or malignant tumor (MPNST) cell lines. Two classes of NFSCs are identifiable, with Class 1 NFSC (green bar beneath the heatmap) expression levels attenuated relative to Class 2 NFSC (blue bar beneath the heatmap) levels. Genes in clusters Cl and C4, which show decreased or increased expression in benign tumors, respectively, and opposite expression in MPNST cell lines (indicated with asterisks), are abundant in genes associated with cell cycle (AURKA, CDC25B, CDKN2A, CNAPl, INHBA, MCM7, PDGFB) and cell differentiation (ADAMl 2, ANGPTL4, BMPl, CHLl, ILIl, INHBA, PPL, SERPINE2). The bar to the right of the heatmap shows five clusters (Cl - C5), corresponding to k-means functional clusters listed in Supplemental Tables 2 and 3.

[00303] In a heat map of transcripts similarly expressed in NFl cell cultures and tumors (figure not shown), differentially expressed transcripts in NFl peripheral nerve cell culture samples were filtered to identify genes with similar patterns of expression in solid tumors. A total of 1,708 transcripts (60%) were identified and clustered across NHSCs, dNFSCs, pNFSCs, MPNST cell lines (annotated as in Figure 1), and dermal neurofibromas (dNFs), plexiform neurofibromas (pNFs) and MPNSTs. The bar to the right of the heatmap shows five clusters (C6 - CI l), corresponding to k- means functional clusters listed in Supplemental Table 5.

[00304] Chromosomal region gene expression patterns in NFl tumors (data not shown) show (a) Intersection of the significant results for hypothesis tests (i) MPNSTs versus dermal and plexiform neurofibromas and (ii) MPNST cell lines versus dermal and plexiform Schwann cells. The blue bands indicate regions where MPNST samples are under-expressed with respect to dermal and plexiform Schwann cell and neurofibroma comparisons. The red bands indicate regions where MPNST samples are over-expressed relative to dermal and plexiform neurofibroma Schwann cells and neurofibromas, (b) High resolution map of putative gains (red) and losses (blue) on human chromosome 17. Genes lost on chromosome 17 encompass p53 and NFl (indicated by white lines). The block of genes over-expressed on the q-arm of chromosome 17 includes SOX9 (indicated by white line). Genes are designated in green, probe set coverage in aqua. The putative gains and losses are designated on both plus (+) and minus (- ) strands.

[00305] Each cluster was re-clustered across NHSCs, primary neurofibromas and primary MPNSTs. We identified sub-cluster(s) that were similarly expressed across cell cultures and their respective solid tumor type. After assigning genes using k-means clustering (Supplemental Table 5), we evaluated clusters C6 - Cl 1 to explore the potential biological significance of transcripts dysregulated in both NFl tumor cell cultures and solid tumors (Supplemental Tables 5 and 6). Unsupervised hierarchical cluster analysis of the transcripts (n = 1,708; 1,108 unique genes) that were similarly expressed in cell cultures and solid tumors again failed to segregate dermal and plexiform neurofibroma tumors (not shown). The complete list of genes is shown in Supplemental Table 4.

[00306] Analysis of functional enrichment for genes in clusters C6 - C9 showed significant associations with nervous system development. Clusters ClO and CI l were associated with skeletal development, and CI l with morphogenesis. Additional functional annotation categories are shown in Supplemental Tables 5 and 6. We used GATACA, a web accessible database of literature-based gene associations to identify genes within each cluster based upon association with nerve development and/or neurofibroma or MPNST. Cluster C6, transcripts downregulated in all sample types, contained EMP2, EPB41L3, GFAP, HLA-DQBl, KLK6, LlCAM, LGIl, MBP and NGFR. Cluster Cl, genes downregulated in MPNST cell lines, MPNSTs, most neurofibromas and variable in Class 2 NFSCs, included CDKN2A, CTSD, GJBl, GNAI2, HPCALl, KNS2, MFl 2, NES and NFKBl. Clusters C8 and C9 exhibited decreased and increased expression, respectively, in MPNST cell lines and MPNST samples. Cluster C8 contained transcripts that included BCL2, BCL2L2, EDNRB, ERBB3, MPZ, PDGFA, PDGFB and SlOOU. Cluster C9 contained transcripts that included EN2, HGF, MDK, PAX6, SMAD3 and WTl. Genes in cluster ClO, variably upregulated in Class 1 NFSCs and upregulated in all others, included APOD, CASPl, CD36, EGFR, KIT, LEPR, MME, and SOCS3. Cluster Cl 1, composed of transcripts including ADM, CAPNl, FBN2, IGFBP 3, PDGFRA, PIAS3, PLAU, PTGES, PTGS2 and TFPI, as well as the neural crest markers TWISTl and SOX9, exhibited increased expression in all samples.

[00307] NFl tumor samples display transcriptional imbalance

[00308] Contiguous chromosomal regions containing multiple over- or under- expressed genes can be indicative of genomic structural abnormalities. A chromosomal analysis to detect transcriptional imbalances expression dataset was conducted using the LAP method (Callegaro et al., 2006). Comparison between dNFSCs and pNFSCs versus NHSCs, and dNFs and pNFs versus NHSCs failed to identify any chromosomal regions showing gains or losses (FDR < 0.05). In contrast, when we analyzed MPNSTs versus dNFs and pNFs, and MPNST cells versus dNFSCs and pNFSCs, we identified candidate regions for potential gains or losses that are shared across the cell culture and tumor comparisons (FDR < 0.05) (Supplemental Table 7).

[00309] Chromosomes 3, 11 and 17 show long stretches of imbalance, interpreted as loss. On chromosome 17, the region of loss included NFl and the region of gain included SOX9. However, SOX9 was not amplified in a subset of plexiform neurofibromas using semi-quantitative DNA-PCR of samples with > 5-fold increase in SOX9 expression (data not shown), suggesting a positional effect on transcription rather than gain of gene copy number. TP53113 and P53AIP1, both p53-related genes, are included in the regions of potential loss shown on chromosomes 17 and 11, respectively. TWISTl is included in the putatively amplified region on chromosome 7. Other putative tumor suppressor deletions and oncogene amplifications in MPNSTs are shown in Supplemental Table 8.

Supplemental Table 8. Putative tumor suppressor losses and oncogene gains in MPNST vs. neurofibroma

Putative Tumdr Suppressor deletions in MPNST vs. Neurofibrbrήa

[nterva "lV start iif era' fqf Genes in interval)

67784234 68784234 DIRAS3

77743223 78743223 DNAJB4

86162412 87870245 CLCA2

99384185 100925066 CDC14A

9265704 10765595 OGGl, TADA3L

19463762 20496457 PCAF

86859286 87859286 POUlFl

ADPRH, CASR, EAF2,

119875114 125313834 GSK3B

117677371 119490705 SLC18A2

10

122992615 124758418 DMBTl

11441120 12765022 DKK3

56400818 58646720 CTNNDl

CCNDl, CDK2AP2, CST6,

58660769 70226916 GSTPl, HRASLS3, MENl,

11 SYVNl

109305803 112043304 PPP2R1B, SDHD

112285527 114315386 NCAMl

115705833 120978584 H2AFX, MLL, POU2F3

123194368 126231305 CHEKl, FEZl, LOH11CR2A

24652490 28502284 KRAS

12

118632639 123374914 PSMD9

[00310] NFl tumor cell culture and solid tumor expression patterns show broad dysregulation of genes activated in developing Schwann cells

[00311] The high representation of genes associated with Schwann cell development in the NFl tumor signature led us to compare the 1,708 gene signature (1,108 unique genes) to gene orthologs activated in neural crest cells and four five stages of Schwann cell development: migrating neural crest, Schwann cell precursor, immature Schwann cell and fully differentiated nerve based on published data sets (Buchstaller et al., 2004; Su et al., 2004) (Table 1). Strikingly, all downregulated clusters (C6 - C8) showed significant enrichment for gene orthologs activated in both immature Schwann cells and fully differentiated nerve. Genes upregulated in MPNST (cluster C9) demonstrated significant over-representation of gene orthologs activated in migrating neural crest cells. Genes over- expressed in all samples but variable in Class 1 NFSCs (cluster ClO) showed enrichment of gene orthologs upregulated in Schwann cell precursors. The repression of transcripts normally expressed late in Schwann cell development and activation of genes normally expressed early is consistent with significant over-representation of developmental themes identified in our functional analysis (Supplemental Tables 4 and 5).

[00312] In cluster Cl 1, we identified the neural crest markers TWISTl and SOX9.

TWISTl inhibits MPNST cell chemotaxis (Miller et al., 2006). A number of SOX family members are differentially expressed in NFl samples relative to normal Schwann cells, including down-regulation of SOX5 (clusters C6 and Cl), SOX2, SOX2OT, SOX8, SOXlO and SOX 13 (cluster C8), and up-regulation of SOXIl (cluster C9) and S0X9 (cluster CI l).

[00313] For technical and biological validation, we measured expression of 82 genes in at least one sample from each sample type by quantitative real time PCR (QRT-PCR). Differential expression of 41 / 82 genes was confirmed in all samples and 77 / 82 genes in at least 50% of the samples (Supplemental Table 9). This confirmation rate is comparable to that reported in other studies. (Rajeevan et al., 2001). The entire dataset will be made publicly available via Gene Expression Omnibus upon manuscript publication.

[00314] SOX9 is over-expressed in NFl-derived peripheral nerve tumors

[00315] SOX9 is a neural crest transcription factor required for stem cell survival

(Cheung et al., 2005). Our microarray data show over-expression of SOX9 in all NFl tumor samples, ranging from 1.5- to 137-fold on the microarray. Average SOX9 expression values were at least two-fold higher in MPNST samples relative to neurofibroma samples. Technical confirmation of SOX9 was conducted by QRT-PCR, using cDNA synthesized from RNA samples used to generate probes for microarray hybridization. SOX9 over-expression was then validated at the RNA and protein levels in independent cultures of human neurofibroma Schwann cells and MPNST cell lines.

[00316] SOX9 is over-expressed in NFl tumors relative to Schwann cells, (a)

Boxplot of SOX9 gene expression microarray data in each of 7 sample types normalized to Schwann cell gene expression (NHSC). Horizontal lines in each bar represent the median SOX9 expression within each sample type and the error bars indicate the range of non-outlier , measurements, (b) SOX9 mRNA expression measured by QRT-PCR of individual samples within each sample type and normalized to expression in Schwann cells (NHSC). Fold-change values were transformed to loglO scale in order to display MPNST samples (ranging from 1448- to 172,950- fold) and NF samples (ranging from 20- to 6,654-fold) on the same graph. Technical validation of RNA samples that were analyzed by microarray include: 1 - 3 = NHSCs; 4 = dNFSC+/-; 5 = dNFSC-/-; 6 = dNF; 7 = pNFSC; 13 = pNF; 14, 22 = MPNSTs. Biological validation of independent RNA samples include: 8 - 12 = pNFSCs; 15 - 21 = MPNST cell lines; 15 = STS26T; 16 = ST8814; 17 = S462; 18 = T265; 19 = 90-8; 20 = 88-3; 21 = YSTl. (c) SOX9 protein expression in Schwann cells (NHSC), MPNST cell lines (26T, 8814, S462, T265) and plexiform neurofibroma Schwann cells (NFSC) by Western blot (top panel). In the experiment, beta-actin was used as a loading control. (d,e) Immunohistochemical detection of SOX9 in a representative neurofibroma (NF) and MPNST (MPNST) tissue section with extensive SOX9 expression. Cells stained brown are positive for SOX9. (e) The distribution of percent SOX9-positive cells in a panel of dermal neurofibroma, plexiform neurofibroma and MPNST sections. The majority of samples screened were represented in the microarray analysis with the addition of 10 independent samples, including 3 dermal neurofibromas, 5 plexiform neurofibromas and 2 MPNSTs. (f) Immunofluorescent detection of SOX9 (TRITC = red) and nuclei (DAPI = blue) in cultured normal human Schwann cells (NHSC), neurofibroma Schwann cells (NFSC) and ST8814 NFl -derived MPNST cells (MPNST). Similar results were obtained in STS26T sporadic MPNST cells (data not shown). Fields shown are representative of each population. Merging fluorescent images detecting DAPI and TRITC (Merge) highlights nuclear localization of SOX9, mainly in MPNST. [00317] Immunohistochemical analysis of SOX9 protein expression was conducted in a panel of 42 NFl tumor sections, 10 of which were independent of the gene expression microarray experiment (data not shown). Strikingly, SOX9 expression was detected in all the tumors. In the majority of dermal neurofibromas, < 30% of the cells were SOX9- positive. The majority of plexiform neurofibromas contained < 50% SOX9-positive cells, while > 70% of the cells were SOX9 positive in the MPNSTs. Concentration of SOX9 in the nucleus was most prominent in MPNST cells, variable in neurofibroma Schwann cells and excluded from the nucleus in normal Schwann cells.

[00318] Reducing SOX9 expression inhibits MPNST cell survival

[00319] To test for a role for SOX9 in tumorigenic cellular behavior, we used shRNAs to reduce SOX9 expression in neurofibroma and MPNST cells (data not shown) relative to a non-targeting control. Decreasing SOX9 expression significantly reduced MPNST cell survival, correlating with an increase in cell death while shRNAs directed toward five other genes (data not shown) did not affect MPNST cell survival. Reducing SOX9 expression with shRNA to a level obtained in MPNST cells had a milder effect on neurofibroma Schwann cell survival, and little or no effect on normal Schwann cells (data not shown). The reduced effect of SOX9 expression in neurofibromas is not solely a function of passage number, as neurofibroma Schwann cells expressing SOX9 shRNAs for several passages showed only a 10 - 20% reduction in growth rate. These data suggest that MPNST cells, not neurofibroma cells, are critically dependent upon SOX9 expression for survival.

[00320] Reducing SOX9 expression in NFl tumor cells inhibits survival, (a)

Confirmation of reduction in S OX9 RNA expression in neurofibroma Schwann cells, (b) Neurofibroma Schwann cells infected with lentivirus expressing SOX9 shRNA or non-specific shGFP control were plated 7 days post-selection in puromycin to measure cell survival in 4 days using a MTS assay. A trend toward a decrease in cell survival was observed in shSOX9-expressing cells compared to shGFP-expressing cells but was not statistically significant. Corresponding phase contrast images to the right show neurofibroma Schwann cells infected with shGFP (NFSC shGFP) or SOX9 shRNA (NFSC shSOX9). (c) Confirmation of reduction in SOX9 RNA expression in MPNST cells by QRT-PCR and (d) SOX9 protein expression by Western blot. Expression levels were measured 7 days post- selection in puromycin and normalized to the non-specific shGFP control. Expression of beta-actin was used as a control, (e) Reduction in MPNST cell number (18-fold) in the presence of SOX9 shRNA relative to shGFP control. Cells were plated in triplicate and represent three independent infections (* p = 0.05). Top right panel represents confluent dish of MPNST cells infected with shGFP lentiviral particles 3 days post- selection with puromycin; bottom right panel represents dying MPNST cells infected with shSOX9 lentiviral particles 3 days post-selection with puromycin. Similar results were observed with 3 different SOX9 shRNAs. (f) MPNST cells were infected with shGFP or shSOX9 lentiviral particles for MTS analysis of cell accumulation during a time course of 1 to 4 days post-selection in puromycin. Cells were plated in triplicate in the presence or absence of puromycin to account for infection efficiency. All shRNAs infect with similar efficiency at greater than 90%. Values are corrected for infection efficiency and represent 3 independent infections, (g) MPNST cells treated as described in (d) and assayed for apoptosis by TUNEL staining 3 days post-selection in puromycin. Uninfected and shGFP- infected cells have similar numbers of TUNEL-positive cells with a significant increase in TUNEL-positive cells in the presence of shSOX9 (** p = 0.002).

[00321] Transcripts differentially expressed in NFl tumor cell cultures and solid tumors are enriched for SOX9 target genes

[00322] SOX9 transcriptional targets described in the literature for other cell types were not significantly altered in our dataset (Panda et al., 2001). However, an analysis of SOX9 binding sites (Mertin et al., 1999) in the promoters of 1,108 unique genes in our NFl tumor signature revealed > 2-times as many potential SOX9 targets relative to the entire genome than expected by chance (39 transcripts as compared to the predicted 18, Fisher's exact

-06 test p = 8.45 ; Supplemental Table 10). The enrichment of SOX9 targets in our dataset is consistent with a role for SOX9 in NFl .

[00323] SOX9 expression throughout the Schwann cell lineage is inferred from work in species other than human. Red represents high SOX9 expression, yellow low SOX9 expression. (1) Dermal and plexiform neurofibroma cells express intermediate levels of SOX9 and show gene signature characteristic of Schwann cell progenitors/immature Schwann cells. (2A) A neural crest gene signature is characteristic of MPNST cells, and neural crest cells are known to express higher levels of SOX9 than more mature cells. (2A, 2B) Neural crest cells may give rise directly to MPNST, or MPNST may form indirectly via a neurofibroma-like cell intermediate.

Supplemental Table 10. Sox9 target genes in transcripts similarly differentially expressed in Schwann cell cultures and tumors

[00324] Each of the k-means clusters identified in Figure 2 (C6 - CI l) was evaluated for over-representation of gene orthologs characteristic of four stages of Schwann cell development. We first generated ortholog gene lists for transcripts upregulated in migrating neural crest, Schwann cell precursors, immature Schwann cells and fully differentiated nerve (Buchstaller et al., 2004; Su et al., 2004). After correcting for redundant gene identifiers and microarray platforms, Fisher's exact test was used to calculate the expected overlaps between k-means clusters and each of the ortholog gene lists. All down regulated clusters (C6 - C8) showed significant enrichment for gene orthologs activated late in Schwann cell development (i.e., immature Schwann cells and fully differentiated nerve). Upregulated gene clusters characteristic of MPNST were C9, enriched for gene orthologs activated early in Schwann cell development (i.e., migrating neural crest), and ClO, enriched for gene orthologs upregulated in Schwann cell precursors. Gene orthologs activated late in Schwann cell development (i.e., fully differentiated nerve) were also significantly over- represented in cluster ClO.

[00325] Example 2.

[00326] Inhibition of Eyes Absent Homolog 4 expression

[00327] The malignant peripheral nerve sheath tumor (MPNST) is the malignant counterpart to benign soft tissue tumors such as neurofibromas and schwannomas. It is most common in the deep soft tissue, usually in close proximity of a nerve trunk. The MPNST may be classified into three major categories with epithelioid, mesenchymal or glandular characteristics. Some of the MPNST include but not limited to, Subcutaneous malignant epithelioid schwannoma with cartilaginous differentiation, Glandular malignant schwannoma, Malignant peripheral nerve sheath tumor with perineurial differentiation, Cutaneous epithelioid malignant nerve sheath tumor with rhabdoid features, Superficial epithelioid MPNST, Triton Tumor (MPNST with rhabdomyoblastic differentiation), Schwannoma with rhabdomyoblastic differentiation. Rare MPNST cases contain multiple sarcomatous tissue types, especially osteosarcoma, chondrosarcoma and angiosarcoma.

[00328] MPNST are aggressive sarcomas without effective therapeutics.

Bioinformatics was used to identify potential therapeutic targets. Expression of a transcriptional network of genes involving Eyes Absent (EYA), Dachshund (DACH), and Sine Oculis (SIX) was dysregulated in MPNST cell lines and solid tumors. EYA proteins are transcriptional activators whose function is modulated by interaction with DACH and/or SIX DNA-binding proteins. We identified a decrease in DACHl expression, and increases in expression of EYAl, EYA2, EYA4, and SIXl- 4. Half of MPNSTs develop subsequent to NFl mutation, and expression of the NFl-GAP related domain (GRD) normalized DACHl expression. EYA4 mRNA was elevated more than 100-fold by quantitative real time PCR in most MPSNT cell lines. In vitro, decreasing EYA4 expression using shRNA reduced cell adhesion and migration and caused cellular necrosis without affecting cell proliferation or apoptotic cell death. MPNST cells expressing sh-EYA4 failed to form tumors in nude mice or formed very small tumors with extensive necrosis but similar levels of proliferation and apoptosis as control cells. The data support EY A4 as a possible target for therapy in MPNST.

[00329] One pathway implicated in MPNST formation is the NF 1 -Ras pathway.

The NFl gene product, neurofibromin, is one of a family of GTPase activating proteins (GAPs) that accelerates the hydrolysis of active Ras- GTP to inactive Ras-GDP (McCormick 1995). Single missense mutations in the GAP-related domain (GRD) of NFl have been detected in patients (Klose, Robinson et al. 1998). Furthermore, neurofibroma Schwann cells and MPNST cell lines and tumors have elevated basal Ras-GTP (Basu, Gutmann et al. 1992; DeClue, Papageorge et al. 1992; Kim, Rosenbaum et al. 1995; Sherman, Atit et al. 2000).

[00330] To identify additional dysregulated molecular pathways in MPNST that might guide novel therapeutic strategies, we conducted global gene expression analysis of NFl -associated tumors and tumor Schwann cells, including sporadic and NFl -derived MPNSTs, on the whole-genome Affymetrix platform (Miller, Jessen et al., 2009). Clusters of genes distinguished MPNST cell lines and primary tumors from neurofibroma Schwann cells, neurofibromas, and normal Schwann cells. The mRNA encoding the S0X9 transcription factor was over-expressed in benign MPNST precursor lesions (neurofibroma), and expression of SOX9 was further elevated in MPNST. EYA4 was identified as a potential transcriptional target of SOX9.

[00331] EYA proteins are the putative effectors of the EYA/SIX/DACH complex, because they contain a highly conserved transcription activation domain and a phosphatase domain that is capable of regulating transcriptional activity of the complex.

[00332] Adenoviral infection. The NFl-GRD adenovirus was constructed by inserting a -1.1kb Xba/HindIII fragment containing the coding sequence for the human NFl-GRD isoform I (GenBank NM_000267; (Viskochil and Carey 1992) and a 5'-HA tag into the pAdTrack vector (He, Zhou et al. 1998). The resulting vector, pAdTrack-NFl -GRD-HA, was linearized with Pmel and transformed into B J5183 cells containing the pAdEasy vector. Recombinants were screened by restriction digestion, sequence- verified, and then transformed into DH5α cells for amplification. Amplified clones were verified by HA expression and ability to reduce Ras activity (see below).

[00333] For microarray and QRT-PCR experiments, MPNST cells were infected with an adenoviral construct expressing the NFl-GRD or GFP, in triplicate, with cesium-purified adenovirus for 2 hours in serum free medium at 200pfu/cell. Viruses were washed away and cells cultured in normal medium for another 30 hours, then RNA isolated as above then RNA was isolated for analysis of gene expression as described (Miller and Jessen et al, 2009). [00334] Lentiviral infection. For lentiviral shRNA infection, we infected

MPNST cells at 70 - 90% confluence with lentiviral particles containing shRNAs targeting EYA4 (Open Biosystems; TRC library) or GFP (Addgene). The CCHMC Viral Vector Core produced virus using a 4- plasmid packaging system. We incubated lentiviral particles with the MPNST cells (MOI ~ 10) in the presence of polybrene (8 μg/mL; Sigma) for 24 hours followed by selection in puromycin at a concentration (2 μg/mL) that killed uninfected cells within three days.

[00335] Ras activation assay. We measured Ras activity in MPNST cells as described (Mahller, Rangwala et al. 2006). MPNST cell lines were infected with the NFl-GRD adenovirus or a vector encoding green fluorescent protein. Two days later lysates were prepared and blotted directly for total canonical Ras proteins (H,N,K-Ras), active ERK, or active MEK. Ras-GTP was evaluated by centrifuging beads conjugated with GST-Raf-RBD from cell lysates, and probing by Western blots (Ras Activation Assay; Upstate Biotechnology).

[00336] Bromodeoxyuridine (BrdU) Incorporation. Twenty-four hours post- plating 3x10⁴ MPNST cells onto glass coverslips, cells were labeled for one hour with BrdU labeling reagent, fixed in ethanol, and incubated with anti-BrdU antibodies followed by detection with fluorescein-conjugated secondary antibodies according to the manufacturer's protocol (5-Bromo- 2'-deoxy-uridine Labeling and Detection Kit II; Roche Applied Science). All cell nuclei were labeled with propidium iodide (PI; 50 μg/mL). Total number of cells (PI+) and number of BrdU positive cells were counted in five fields per sample and averaged.

[00337] Migration assay. The migratory response of MPNST cells was measured using a modified Boyden chamber assay as described (Miller, Rangwala et al. 2006). MPNST cells (4 x 10⁴) expressing shGFP or shEYA4 were plated in serum-free DMEM on the upper chamber of a transwell with 8 μm pores (Costar); The lower chamber contained 800 μl DMEM plus serum. Cells were incubated for 16 h at 37°C in 10% CO₂. Nonmigrating cells were removed from the upper surface of the membrane with cotton swabs. Membranes were stained with bisbenzimide and mounted onto glass slides. Migration was quantified by counting cells in four fields. Each condition was performed in triplicate, and the number of migrated cells was normalized to the total number of cells on an unscraped filter to validate the total number of cells plated. Data shown are representative of three independent experiments; values presented are the mean ± s.d. Statistical significance was determined by t-test using Microsoft Excel software.

[00338] Mouse xenograft. Mouse xenografts of MPNST cells were conducted as described (Mahller, Vaikunth et al. 2007). Approximately one million S462TY MPNST-shControl, MPNST-shEYA4.3, MPNST-shEYA4.5, MPNST-shEYA4.7 were injected subcutaneously into 6- to 8-week-old female athymic nude (nu/nu) mice (Harlan, Indianapolis, IN). In accordance with CCHMC rodent tumor guidelines, tumors may not exceed 10% of body weight. Tumors formed in shControl cells in approximately 30 days, reaching -2000 mm3 in approximately 60 days. Therefore, 59 days post-injection, we measured tumor volume and euthanized all shControl mice and shEYA4.5 mice. Tumors were dissected and submitted to the CCHMC Pathology Lab for histological characterization, including H&E staining, Mib-1 detection of proliferating cells, and TUNEL, CD31.

[00339] Results. [00340] A novel transcription complex is dysregulated in MPNST.

[00341] Global gene expression profiling in NFl -associated tumors and Schwann cells revealed a cluster of genes significantly over-expressed in MPNSTs relative to neurofibromas and normal Schwann cells (Miller and Jessen, et al., 2009). The most striking observation within this cluster was an up- regulation in expression of EYA4 and one of its potential binding partners, SIXl. EYA, SIX, and DA CH proteins function downstream of PAX6 in a transcriptional complex, and the transcriptional regulator of EYA and SIX genes, PAX6, was also upregulated in MPNST cells. Furthermore, the E Y A/SIX binding partner DACHl was downregulated. Expression profiles of EYA, SIX, and DACH genes show differential expression of multiple family members in more than one MPNST sample relative to normal Schwann cells, including down-regulation of DACHl and up- regulation in EYAl, EYA2, EYA4 and SIX1-4. DACHl expression is lower than normal in most benign neurofibroma samples as well as MPNST samples. Expression levels of EYA4 and SIXl are statistically different between benign neurofibromas and MPNSTs and up-regulated at least three-fold in MPNST samples (data not shown).

[00342] Expression of the P AX/E YA/SIX/D ACH transcriptional complex is dysregulated in MPNST. (A) Hierarchical clustering of gene expression microarray data of PAX, EYA, SIX, and DACH genes family members. EYA4, SIXl, and PAX6 are statistically distinguished (ANOVA; FDR= Ol) between normal Schwann cells, neurofibroma samples, and MPNST samples and expressed, on average, at least three-fold higher in MPNST samples (n=6 primary MPNSTs; n=14 MPNST cell lines) relative to neurofibroma samples or normal Schwann cells, yellow = normal; red = overexpression; blue = underexpression. DACHl expression is down- regulated in MPNST cell lines and the majority of benign neurofibroma samples. (B) Confirmation of microarray gene expression data for PAX6, SIXl, EYA4, and DACHl using quantitative real time PCR (QRT-PCR). Black bars = microarray data; White bars = QRT-PCR data. Values are fold-change in expression relative to normal human Schwann cells (N). Samples 1 - 8 are MPNST cell lines: 1 = STS26T; 2 = ST8814; 3 = S462; 4 = T265; 5 = S520; 6 = 90-8; 7 = 88-3; 8 = YSTl.

[00343] We confirmed the gene expression microarray data revealing differential expression of PAX6, SIXl, EYA4, and DACHl in MPNSTs relative to NHSCs using quantitative real-time PCR (QRT-PCR) (data not shown). The magnitude of differential expression obtained using QRT-PCR was higher as compared to the microarray results yet displayed the same trend. The QRT-PCR data corroborated the most robust overexpression in EYA4, ranging from 50-fold to over 800-fold above NHSC. The microarray expression data for DACHl was somewhat variable in normal Schwann cells (ranging from -3.7 to 3.2-fold relative to the mean), and one MPNST cell line, YSTl, displayed overexpression of DACHl. QRT-PCR results validated the microarray data, confirming reduced DACHl expression in all MPNST cell lines, with the exception of YSTl, relative to 3 independent normal Schwann cell samples.

[00344] The NFl-GAP related domain (GRD) regulates expression of DACHl in MPNST cells. The NFl-GRD is the best characterized domain of NFl, converting activated Ras to its inactivated form. To observe transcriptional changes downstream of the NFl-GRD, we conducted gene expression microarray analysis on MPNST cells 32 hours after infection with an adenovirus encoding the NFl-GRD elative to a GFP control (Data not shown), a time point representing significant reduction in Ras-GTP and prior to cell death (Data not shown). Forty five genes were differentially expressed (>3-fold) in the MPNST 8814 cell line response to the NFl-GRD (data not shown), including DACHl. Three of six DACHl probe sets confirmed the decrease in expression in ST8814 MPNST cells relative to normal human Schwann cells that was not significantly changed by infection with a control adenoviral vector but increased significantly subsequent to infection with the NFl-GRD adenovirus. Failure to show this expression pattern with three DACHl probe sets was likely due to probe set unreliability (Dai, Wang et al. 2005). Expression of PAX, EYA, and SIX genes was not significantly altered (Data not shown), but DACHl expression was increased to a level comparable with normal human Schwann cells (Data not shown).

[00345] Importantly, we confirmed that MPNST cells infected with the NF 1 -GRD adenovirus have significant reductions in Ras-GTP and downstream phospho-MEK and phospho-ERK protein levels at the 32 hour time point (Data not shown). In addition, the significant increase in DACHl expression by the NFl-GRD adenovirus was not observed with the GFP adenovirus control (Data not shown). These results were confirmed by time course QRT-PCR, showing a progressive increase in DACHl expression levels from 18 - 32 hours in MPNST cells infected with the NFl-GRD relative to the GFP control (Data not shown).

[00346] The NFl-GRD normalizes DACHl expression. (A) Gene expression microarray data of normal human Schwann cells (NHSC), and ST8814 NFl patient-derived MPNST cells (Control) infected with an adenoviral vector expressing GFP (Vector) or the NFl-GRD (GRD). Samples were analyzed in triplicate, and expression is relative to the mean of the NHSC samples. Yellow = normal; Red = overexpression; Blue = underexpression. The microarray expression data for three DACHl probe sets (*) is expressed as fold-change relative to NHSC in the bar graph to show normalization of DACHl expression by the NFl-GRD. (B) The NFl-GRD adenovirus blocks Ras/Map-kinase signaling in ST8814 MPNST cells. Ras-GTP was measured in cell lysates as a measure of efficacy, inhibition of Ras signaling. Ras-GTP, phospo-MEK and phosphor-ERK protein levels were diminished in the presence of the NFl- GRD. (C) Microarray gene expression relative to uninfected ST8814 MPNST control cells is significantly increased by the NFl-GRD. (D) Confirmation of increased levels of DACHl expression in response to the NFl-GRD at 18, 24, and 32 hours post-infection using QRT-PCR. Values are fold-change in expression relative to adenoviral vector control.

[00347] Reducing EYA4 expression inhibits MPNST cell adhesion and migration. To study the functional role for EY A4 in MPNST tumorigenesis, we first validated the microarray and QRT-PCR data by confirming elevated levels of EYA4 protein in MPNST cell lines relative to NHSCs at the protein level by Western blotting. Two bands were observed in MPNST cell lines, likely representing the products of EYA4 transcript variant 1 and transcript variant 2 (Borsani, DeGrandi et al. 1999), but not in NHSCs (Data not shown).

[00348] To observe the effects of reducing EYA4 expression in MPNT cells, stable

S462TY MPNST cell lines expressing EYA4 shRNAs were created. We observed a marked decrease in EYA4 expression at the RNA and protein levels using 4 independent EY A4 shRNAs compared to the scramble shRNA control (Data not shown). Similar results were obtained in 2 additional independent MPNST cell lines (data not shown). The extent of EY A4 knockdown varied from 2 to 9-fold and was stable, as the pattern of expression across clones was retained through several cell passages (pi - p6). [00349] Reducing EY A4 expression with shRNA inhibits cell adhesion and migration. (A) Confirmation of expression of EYA4 in MPNST cell lines (STS26T, ST8814,S462,T265) and not normal human Schwann cells (NHSC) at the protein level by Western blotting. Actin protein levels confirmed equal amounts of protein across samples. (B) Confirmation of reduction in EY A4 expression in S462Ty MPNST cells (control) subsequent to introducing independent shEYA4 RNAs (4.3,4.4,4.5,4.7) at the RNA level using QRT-PCR and protein level by Western blotting. Pictures of live cells at day 0 (dθ) and day 4 (d4) exhibit rounded morphology changes indicative of adhesion changes in S462TY cells containing shEYA4 shRNAs. (C) Cells remaining after wash in an adhesion assay are significantly reduced (*p<0.05) in the presence of EY A4 shRNAs (4.3, 4.7) relative to control shRNA. (D) Migration of S462TY MPNST cells is significantly reduced (*p<0.05; **p<0.01) in the presence of EYA4 shRNAs (4.3, 4.4, 4.5). Values are expressed as percent migration relative to control shRNA.

[00350] To test the effect of reducing EY A4 on specific cellular mechanisms that regulate MPNST cell behavior, we conducted functional assays in vitro. The rounded appearance of cells expressing EYA4 shRNA suggested a defect in cell adhesion that was confirmed in a cell adhesion assay (Data not shown). When EYA4 expression is reduced in MPNST cells, a 60 - 95% reduction in migration was evident (Data not shown). Inhibition of migration correlated with reduction in EY A4 expression. Thus, cell adhesion and motility are dependent on EYA4.

[00351] Reducing EY A4 expression induces MPNST cell death by necrosis.

An apparently slower growth rate was observed in culturing the MPNST- shEYA4 cells. A modest decrease in cell accumulation was evident, yet not significant, in a nine day MTS assay (data not shown). Further investigation by FACS analysis confirmed no significant change in cell proliferation, apoptosis, or cell cycle progression. BrdU incorporation was used to confirm similar rates of proliferation in MPNST-shEYA4 and MPNST-shControl cells. A very small percentage of apoptotic cells were detected in both MPNST cells expressing shEYA4 and shControl as confirmed by TUNEL analysis (data not shown).

[00352] Evidence of cell death by necrosis was observed in MPNST-shEYA4 cells

(Data not shown). Cell death is associated with morphological changes including cell shrinkage, cytosolic condensation, membrane morphology changes, loss of anchorage and finally, cellular degradation (Krysko, Vanden Berghe et al. 2008). By flow cytometry, induction of cell death generally results in a shift of low side light scatter population to higher side light scatter population (Dive, Gregory et al. 1992; Weston, Alsalami et al. 1994; Waring, Lambert et al. 1999). To prevent analysis confusions with possible artifacts from cell aggregates and debris in suspension, our analysis by flow cytometry has focused on cell singlets of adhered cells. We could see that despite performing the flow cytometry analysis in the adherent cell fraction, which is enriched in surviving cells, there was a -10% increase in their light side scatter mean channel (Data not shown).

[00353] Reducing EY A4 expression inhibits MPNST formation ex vivo. To determine if EYA4 expression is critical for MPNST tumorigenesis, we tested the MPNST-shEYA4 cells in a mouse xenograft model using the S462TY cell line. By 60 days, MPNST cells expressing 3 independent EY A4 shRNAs displayed significant reduction in tumor volumes. As seen with the in vitro cell migration studies above (Data not shown), a correlation with EY A4 reduction and tumor volume was observed. Expression levels of EYA4 in MPNST-shEYA4.5 and MPNST-shEYA4.5 were reduced greater than 6-fold and did not develop detectable tumors; Expression levels ofEYA4 in MPNST-shEYA4.3 were reduced only 2- fold and developed small tumors by 60 days. Reduced expression of EYA4 was maintained in the MPNST-shEYA4.3 tumors relative to control tumors, as confirmed by QRT-PCR (Data not shown).

[00354] Histological analyses revealed significant areas of necrosis within the shEYA-expressing tumors. Similar degrees of proliferation and apoptosis in Control and shEYA tumors were confirmed with MIB-I staining and TUNEL assay, respectively. CD31 staining revealed similar vascularity as well.

[00355] Discussion

[00356] Microarray gene expression analysis revealed a cluster of genes differentially expressed in MPNST relative to normal human Schwann cells including members of the SIX-DACH-EYA transcription complex. The NFl-GRD induced DACHl expression implicating Ras signaling in decreased DACHl expression in MPNST. Expression of EYA4 was dramatically upregulated in MPNST cells and primary tumors and carries the transcription activation domain of the complex, making it an attractive candidate for future investigation. Inhibition of EYA4 expression with shRNA reduced MPNST cell adhesion and migration and caused cell necrosis. Tumorigenesis was profoundly inhibited when MPNST cells stably expressing shEYA4 RNA were injected into athymic nude mice, again correlating with necrosis. Thus the EY A4-D ACH-SIX 1 complex is a novel transcriptional pathway dysregulated in MPNST tumorigenesis, blocking cell necrosis.

[00357] PAX6 expression was upregulated in the majority of MPNST cell lines and tumors. MPNST gene expression profiles resemble that of a primitive neural crest-like cell (Miller and Jessen et al., 2009), and PAX6 expression is associated with CNS neural stem cell migration, proliferation, and differentiation (Osumi, Shinohara et al. 2008)and pancreatic (Lang, Mascarenhas et al. 2008), brain and breast cancer cell lines (Muratovska, Zhou et al. 2003). Multiple SIX and EYA family member were changed in expression as compared to normal human Schwann cells. None has been studied in Schwann cells or MPNST previously, and each may play a role in MPNST. However only SIXl, EY A4, DACHl were significantly changed and it is likely that these are the relevant proteins in MPNST cells. SIXl was at least modestly over-expressed in all MPNST cells and tumors relative to normal Schwann cells, and expression of SIXl has been implicated in initiating and promoting tumorigenesis in other tissue types; SIXl promotes tumor cell proliferation, migration, invasion, and metastasis (Christensen, Patrick et al. 2008). Conversely, DACHl expression was down-regulated in MPNST cells relative to normal Schwann cells. Consistent with our results, loss of DACHl is low in breast and prostate cancer cells and restoring DACHl expression blocks tumorigenic properties (Wu, Li et al. 2006; Wu, Katiyar et al. 2008; Wu, Katiyar et al. 2009). EYA4 was robustly overexpressed in all but one MPNST cell line and all primary MPNSTs, suggesting it may represent an MPNST oncogene, inconsistent with reports implicating EYA4 as a tumor suppressor gene in gastrointestinal tumors (Zou, Osborn et al. 2005; Osborn, Zou et al. 2006). EY A4 was up-regulated in NFl -related and in sporadic MPNST lines, even though Ras-GTP is not elevated in these same sporadic MPNST cell lines (Mahller, Rangwala et al. 2006). The mechanism(s) underlying high EYA expression remain to be determined. One such mechanism may be elevated SOX9 expression, as increasing SOX9 in wild type Schwann cells is sufficient to increase EY A4 mRNA expression, while decreasing SOX9 mRNA levels in MPNST cells decreases EYA4 expression (Miller and Jessen et al., 2009). Although DACHl expression was not down-regulated in MPNST primary tumors, the data show that decreased DACHl expression is an early event downstream of NFl loss, because replacement of the NFl-GRD in MPNST cells rescues DACHl expression. The decreased DACHl as an early event in tumorigenesis is demonstrated in that DACHl expression is low in many neurofibroma Schwann cell cultures and primary neurofibromas. In contrast, only some neurofibroma samples have elevated SIXl or EYA4 expression. It is possible that a primary effect of loss of NFl on DACHl secondarily affects transcription of the other members of the complex. Indeed, several lines of evidence support feedback activation of the network members on each other (Rebay, Silver et al. 2005). Replacement of the NFl-GRD did not alter transcription of EYA or SIX mRNAs, but we cannot exclude the possibility that at other time points transcription might be changed. For example, it is possible that prolonged imbalance in the stoichiometry of EYA/SIX/DACH complex due to diminished DACHl favors the transcription of EY A/SIX activation complex, activating EYA and SIX themselves, as well as EYA target genes. Furthermore, in Drosophila it has been shown that Ras signaling phosphorylates EYA proteins to increase transcriptional activation (Hsiao, Williams et al. 2001; Silver, Davies et al. 2003). However, other mechanism(s) must account for deregulation in the absence of NFl mutation. LITERATURE CITED

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Claims

What is claimed is:

1. A method of diagnosing a neurofibromatosis type 1 (NFl)-derived tumor or a predisposition to developing a neurofibromatosis type 1 (NFl)-derived tumor in a subject, comprising determining a level of expression of a NFl -associated gene in a patient derived biological sample, wherein an increase or decrease of the level expression compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing a neurofibromatosis type 1 (NFl)- derived tumor and/or other NFl manifestation.

2. The method of claim 1 , wherein the NF 1 -associated gene is at least one gene selected from the group consisting of the genes listed in Tables 1-3, wherein an increase in the level compared to a normal control level indicates the subject suffers from or is at risk of developing a neurofibromatosis type 1 (NFl)-derived tumor.

3. The method of claim 2, wherein the increase is at least 2 fold than the normal control level and such increase indicates that the subject suffers from or is at risk of developing a neurofibroma, malignant peripheral nerve sheath tumors (MPNST), and/or other peripheral nerve tumor or sarcoma.

4. The method of claim 1 , wherein the NF 1 -associated gene is at least one gene selected from the group consisting of the genes listed in Tables 1, and wherein an increase in the level compared to a normal control level indicates the subject suffers from or is at risk of developing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma.

5. The method of claim 4, wherein the increase is at least 2 fold higher than the normal control level.

6. The method of claim 1 , wherein the method further comprises determining the level of expression of a plurality of NFl -associated genes.

7. The method of claim 1, wherein the expression level is determined by any one method select from group consisting of: (a) detecting the mRNA of the NFl- associated genes, (b) detecting the protein encoded by the NFl -associated genes, and (c) detecting the biological activity of the protein encoded by the NFl- associated genes.

8. The method of claim 1, wherein the level of expression is determined by detecting hybridization of a NFl -associated gene probe to a gene transcript of the patient- derived biological sample.

9. The method of claim 8, wherein the hybridization step is carried out on a DNA array.

10. A neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma reference expression profile, comprising a pattern of gene expression of two or more genes selected from the group consisting of the genes listed in any of Tables 1-3.

11. A method of screening for a compound for treating or preventing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma, the method comprising the steps of: a) contacting a candidate compound with a cell expressing one or more marker genes, wherein the one or more marker genes is selected from the group consisting of the genes listed in Tables 1-3; and b) selecting a compound that reduces the expression level of one or more marker genes selected from the group consisting of the genes listed in Tables 1-3, or elevates the expression level of one or more marker genes selected from the group consisting of the genes listed in Tables 1-3.

12. An array comprising a nucleic acid which binds to two or more nucleic acid sequences selected from the group consisting of the genes listed in Tables 1-3.

13. A method of treating or preventing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma in a subject comprising administering to the subject a compound that increases the expression or activity of the genes listed in Tables 1-

3.

14. A method for treating or preventing neurofibroma, MPNST and/or other peripheral nerve tumor or sarcoma in a subject, the method comprising the step of administering a compound that is obtained by the method according to claim 11.

15. A method for detecting, or for detecting and distinguishing between or among cell proliferative disorders in a subject, comprising determining, in a biological sample isolated from a subject, the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes, respectively.

16. The method of claim 15, wherein an increase in expression level of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIX1-4 genes is determinative of a malignant peripheral nerve sheath tumor (MPNST) cell proliferative disorder in a subject.

17. The method of claim 15, wherein the expression level determined is at least a three-fold increase in expression level of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIXl -4 genes compared to normal cell expression.

18. The method of claim 15, wherein the method further comprises comparing the expression level to a normal standard wherein an decrease in DACHl expression compared to normal expression indicates that the subject is a candidate for further examination for cancer.

19. The method of claim 15, wherein the method further comprises comparing the expression level to a normal standard wherein an increase in expression of one or more of EYAl, EYA2, EYA4, and SIX1-4 compared to normal expression indicates that the subject is a candidate for further examination for cancer.

20. A method for identifying an agent as a candidate for treating cancer, the method comprising the steps of: exposing cancer cells to a test agent; measuring the expression levels of one or more genes selected from the group consisting of Eyes Absent (EYA), Dachshund (DACH), Sine Oculis (SIX) and paired box-containing (PAX) genes; and comparing the expression level to that of control cells not exposed to the test agent wherein a lower or higher than control expression indicates that the agent is a candidate for treating cancer.

21. The method of claim 20, wherein an increase in DACHl expression compared to control expression indicates that the agent is a candidate for treating cancer.

22. The method of claim 20, wherein a decrease in the expression levels of one or more genes selected from the group consisting of EYAl, EYA2, EYA4, and SIX1-4 compared to control expression indicates that the agent is a candidate for treating cancer.

23. A method for determining the effectiveness of a treatment for cancer, the method comprising the steps of: measuring the expression levels of one or more genes selected from the group consisting of DACHl, EYAl, EYA2, EYA4, and SIXl -4 genes in a first sample from a cancer patient prior to providing at least a portion of the treatment to the patient; measuring the expression of the expression levels in a second sample from the patient after the portion of the treatment is provided to the patient; and comparing the expression levels of the first sample and the second sample wherein an increase in DACHl expression and/or a decrease in the expression of one or more of EYAl, EYA2, EYA4, and SIXl -4 expression level in the second sample indicates that the treatment is effective.

24. A method for treating or preventing cancer in a human or non-human animal comprising the step of: administering to the human or non-human animal having cancer an active agent in an amount effective for treating cancer wherein the active agent comprises a therapeutic agent effective to increase DACHl expression and/or decrease expression of one or more of EYAl, EYA2, EYA4, and SIX1-4.

25. The method of claim 24, wherein the active agent further comprises an additional cancer therapeutic agent.

26. A method of a treatment for malignant peripheral nerve sheath tumors (MPNST) comprising administering to a patient in need thereof a biologically effective amount of one or more therapeutic agents capable of directly or indirectly decreasing the expression or activity levels of EYA4.