HK1191100A - A method of analysing a blood sample of a subject for the presence of a disease marker - Google Patents

Abstract

The present invention relates to a method of analyzing a blood sample of a subject for the presence of a disease marker, said method comprising the steps of a) extracting nucleic acid from anucleated blood cells in said blood sample to provide an anucleated blood cells-extracted nucleic acid fraction, and b) analyzing said anucleated blood cells-extracted nucleic acid fraction for the presence of a disease marker, wherein said disease marker is a disease-specific mutation in a gene of a cell of said subject, or wherein said disease marker is a disease-specific expression profile of genes of a cell of said subject.

Description

Method for analyzing a blood sample of a subject for the presence of a disease marker

Technical Field

The present invention is in the field of medical diagnostics, and in particular in the field of disease diagnosis and monitoring. The present invention relates to a marker for detecting a disease, a method for detecting a disease, and a method for determining the effectiveness of a disease treatment.

Background

In clinical practice, there is a great need for therapies that can detect the disease at its earliest stages, can predict the progression of the disease, and can be tailored to the patient. In particular early detection of neoplastic diseases (cancer) plays a crucial role in ensuring a favorable treatment of the disease. Despite the many advances in medical research, cancer remains a leading cause of death worldwide. When patients seek treatment, they often show symptoms of distant metastases, which means that events with too late detection of cancer occur too frequently.

Lung, prostate, breast and colorectal cancers are the most common tumors and a fast and simple method for early diagnosis of cancer is needed to assist surgical resection, radiotherapy, chemotherapy or other known therapies to take appropriate remedial measures. The availability of good diagnostic methods for cancer is also important for assessing patient response to treatment, or for assessing recurrence due to regrowth at the primary site or metastases.

Several types of cancer markers are currently known, such as oncogene products, growth factors and growth factor receptors, angiogenic factors, proteases, attachment factors and tumor suppressor gene products, etc., and are considered essential not only for early diagnosis but also for the following diagnosis: differentially diagnosing patients with indeterminate clinical deformities, e.g., to distinguish between benign and malignant deformities; for predicting the likelihood that a particular patient with a defined malignancy will respond to a therapy selected for treatment; and for providing information about the risk of developing a malignancy, the presence of a malignancy, or future behavior of a malignancy in a human or animal subject. The ability to detect and diagnose cancer by detection of tumor or cancer markers is currently an area of general interest, and there is a need for reproducible and reliable methods of identifying new and more cancer markers in patient samples.

Glioblastoma is the most common and most aggressive type of primary brain tumor in humans, a disease that is difficult to diagnose and even treat, in part because of the blood-brain barrier that hinders the delivery of therapeutic agents and hinders the detection of potentially important diagnostic markers. Diagnostic markers for glioblastoma are available, but are specific for the tumor tissue itself and require a tumor sample.

Improved screening and detection methods are needed to detect cancer at an early stage and to follow the progression of the disease. In the case of cancer, we are in the following states: we need not only to detect a tumor, but also to detect it before it reaches the point of failure to recover (where the treatment becomes palliative rather than curative). People at risk as well as patients with recurrent cancer should be monitored comprehensively. In addition, since tumors may respond differently to different therapies, patient stratification becomes important.

Genetic analysis using tumor biopsies has allowed the identification of many mutations that are useful for cancer diagnosis as well as for visualizing patient stratification strategies. However, the current weakness of tumor inheritance is the need for tumor biopsies, which are often impossible to dissect from the patient. In addition, the use of biopsies is static and does not allow genetic monitoring of the progression or recurrence of a tumor over time. In addition, many tumors are heterogeneous, resulting in the genetic characterization of potential false positives or false negatives for such tumor biopsies.

Recently, the use of circulating tumor cells for diagnosis and monitoring of tumor progression or recurrence has shown the use of blood as a source of tumor-derived material, particularly tissue debris in the form of cells. The use of circulating tumor cells is not effective for most cancers.

Calverley et al (Clinical and Translational Science Vol.3, 5 th, 2010) disclose the down-regulation of platelet gene expression in metastatic lung cancer. The authors identified 200 genes that showed differential expression between healthy persons and patients. As the authors state, the proteome of platelets is reflected in the transcriptome of platelets. Gene expression as measured correlates with genes from megakaryocytes. There is no disclosure that RNA/DNA derived from cells other than megakaryocytes is measured when thrombocytes are tested, and there is no indication that circulating RNA/DNA derived from other cells can be taken up by thrombocytes.

Generally, disease markers are defined as compounds that: the concentration of the compound is altered, preferably increased, in biological fluids from diseased patients when compared to normal healthy subjects, and the compound is then used as a marker compound indicative of disease. However, the recognition of specific compounds, e.g. proteins, as markers for diseases (such as cancer) in various body fluids has been hampered by the lack of suitable techniques.

In the case of other diseases than cancer, markers that are difficult to detect can also be obtained. This hampers early diagnosis of the disease.

Lood et al (Blood volume 116, 11 th, 2010) disclose elevated gene expression of IFN-I regulated genes in platelets of patients with SLE. The authors hypothesized that IFN α affects gene expression in megakaryocytes, resulting in elevated levels of IFN-I regulated proteins in platelets. Thus, gene expression from megakaryocytes is associated with SLE or vascular disease. There is no disclosure that RNA/DNA from diseased cells can be taken up by platelets.

The present invention aims to overcome the problem of the prior art that not all diseased tissues or disease types (e.g. tumours) give rise to circulating diseased cells (e.g. circulating tumour cells). The present invention also aims to overcome the problem of protein markers for detecting diseases such as cancer being difficult to detect. In addition, the present invention aims to provide a method that does not require a biopsy and allows a comprehensive monitoring of the patient.

Disclosure of Invention

In a first aspect, the present invention provides a method of analysing a blood sample of a subject for the presence of a disease marker, the method comprising the steps of: a) extracting nucleic acid from the anucleated blood cells, preferably thrombocytes, in the blood sample to provide an anucleated blood cells extracted nucleic acid fraction; and, b) analyzing the anucleated blood cells extracted nucleic acid fraction for the presence of a disease marker; wherein the disease marker is a disease-specific mutation in a gene of a nucleated cell of the subject; or, wherein the disease marker is a disease-specific expression profile of a gene of a nucleated cell of the subject.

It has surprisingly been found that nucleic acids from nucleated cells are present in anucleated blood cells, such as thrombocytes. This may be the discharge of nucleic acids into the bloodstream by the nucleated cells, and then uptake of these discharged nucleic acids from the bloodstream by the enucleated cells (such as thrombocytes), or the transfer of nucleic acids from the nucleated cells to the enucleated blood cells in some other transport manner. The inventors have for the first time realized that disease markers can be used for nucleic acids extracted from anucleated blood cells to identify diseases from nucleated cells.

In a preferred embodiment of the method of the invention, said anucleated blood cell-extracted nucleic acid fraction comprises nucleic acids originating from nucleated cells. In a preferred embodiment of the present invention and embodiments thereof, said anucleated blood cell-extracted nucleic acid fraction is not a megakaryocyte-derived nucleic acid or a megakaryocyte-derived RNA, i.e.the nucleic acid fraction to be detected is not a megakaryocyte-line or a megakaryocyte-genome-derived nucleic acid fraction.

The term "anucleated blood cells" as used herein refers to cells that lack a nucleus. The term includes erythrocytes and thrombocytes. In various aspects of the invention, a preferred embodiment of an enucleated cell is a thrombocyte. The term "anucleated blood cells" preferably does not include cells lacking nuclei due to erroneous cell division.

The term "nucleated cell" as used herein refers to a cell having a nucleus. The term includes somatic cells, germ cells, and stem cells, and can include cells from colon, pancreas, brain, bladder, breast, prostate, lung, breast, ovary, uterus, liver, kidney, spleen, thymus, thyroid, neural tissue, connective tissue, blood, epithelial tissue, lymph node, bone, muscle, and skin tissue. The nucleated cells are preferably cells from diseased tissue. In a preferred embodiment, the above-mentioned nucleated cells are not megakaryocytes.

Thus, the present invention is generally directed to analyzing nucleic acids that have been transferred from cells with nuclei to cells without nuclei, wherein the cells without nuclei can be easily isolated from the blood stream and contain nucleic acids from the nucleated cells.

The term "nucleus" refers to a membrane-enclosed organelle found in eukaryotic cells that contains a large portion of the cellular genetic material organized in a chromosomal fashion. The genes within these chromosomes are the nuclear genome of the cell. The interior of the nucleus contains many nucleosomes that contain an RNA-containing nucleoli that is primarily involved in the assembly of ribosomes that contain RNA. After production in the nucleolus, ribosomes are exported to the cytoplasm where they translate the mRNA.

The nucleic acid fraction extracted from the anucleated blood cells is preferably a fraction comprising chromosomal DNA, ribosomal RNA, nucleolar RNA and/or messenger RNA.

As used herein, and particularly in the phrase "mutations in genes that nucleate cells," the term "gene" refers to any nucleic acid sequence that nucleates a (somatic) cell, including chromosomal and extrachromosomal nucleic acid sequences, preferably nuclear nucleic acid sequences, and may include transcribed and non-transcribed sequences as well as ribosomal RNA sequences, most preferably chromosomal sequences that are transcribed into RNA.

In a preferred embodiment of the method of the invention, the disease-specific mutation is located in a chromosomal gene.

In another preferred embodiment, the gene is not a gene from a anucleated blood cell. In another preferred embodiment, the gene is not a gene from a megakaryocyte. In yet another preferred embodiment, the gene is not CD 109.

In a preferred embodiment of the method of the invention, the disease-specific expression profile is an expression profile of a chromosomal gene. In particular, mRNA in chromosomal genes from nucleated cells is present in thrombocytes.

In another preferred embodiment of the method of the invention, the nucleic acid is a ribonucleic acid (RNA), more preferably a messenger ribonucleic acid (mRNA).

In a preferred embodiment of the method of the invention, the nucleic acid is not mtDNA. Accordingly, mitochondrial nucleic acids are preferably not an aspect of the present invention.

In another preferred embodiment of the method of analyzing a blood sample according to the present invention, said step b) of analyzing for the presence of a disease marker in said anucleated blood cell-extracted nucleic acid fraction comprises:

i) selectively amplifying the mutation by polymerase chain reaction amplification of reverse transcriptase using at least one nucleic acid mutation specific amplification primer or probe; or

ii) selectively amplifying the plurality of mRNAs by polymerase chain reaction amplification of reverse transcriptase to determine the expression level of a chromosomal gene encoding the mRNA, thereby providing an expression profile of the gene and comparing the expression profile to a reference profile.

The blood sample is preferably in vitro.

In a preferred embodiment of the method of the present invention, the above-mentioned diseases are selected from the group consisting of cancer, autoimmune diseases, skin diseases, eye diseases, endocrine diseases, neurological disorders and cardiovascular diseases.

In another preferred embodiment of the method of the invention, the disease is selected from the group consisting of autoimmune diseases, skin diseases, eye diseases, endocrine diseases, neurological disorders and cardiovascular diseases.

In another preferred embodiment of the method of the invention, the disease is cancer.

In a further preferred embodiment of the method of the invention, the cancer is a solid tumor cancer, preferably selected from colon, pancreas, brain, bladder, breast, prostate, lung, breast, ovary, uterus, liver, kidney, spleen, thymus, thyroid, neural tissue, epithelial tissue, lymph node, bone, muscle and skin.

In another preferred embodiment of the method of the invention, the disease is not cancer.

In another preferred embodiment of the method of the invention, the disease is not a vascular disease.

In another preferred embodiment of the method of the invention, the disease is not systemic lupus erythematosus.

In another preferred embodiment of the method of the invention, the disease is not a sickle cell disease.

In another preferred embodiment of the method of the invention, the disease is not Alzheimer's disease.

In another preferred embodiment of the method of the invention, the disease is not a disease associated with pathological megakaryocyte function.

In another preferred embodiment of the method of the invention, the disease is not a disease associated with pathological platelet function.

The above-described embodiments, which are discarded in the preferred embodiments, may be combined in any combination.

In another preferred embodiment of the method of the invention, the disease is selected from the group consisting of autoimmune diseases, skin diseases, eye diseases, endocrine diseases and neurological disorders.

In a preferred embodiment of each aspect of the invention, the Autoimmune Disease is selected from the group consisting of achlorhydria Autoimmune Active Chronic Hepatitis (Achlorhydra Autoimmune reactive Hepatitis), acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's Disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, anti-synthetase syndrome, polyarthritis, Atopic dermatitis, Autoimmune aplastic anemia,Autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative Syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrinal gland Syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura (autoimmune purpura), autoimmune uveitis, Barlow's disease/Barlow's sclerosis, Behcet's Syndrome, Berger's disease, Bickerstaff's encephalitis, Blauu Syndrome, Bullous Pemphigoid (Bullous Pemphigoid), Cassiemann's disease, Celiac disease (Celiac disease), chronic fatigue Syndrome, chronic functional fatigue Syndrome, autoimmune lymphoproliferative disorder, autoimmune peripheral neuropathy, autoimmune diseases, Chronic inflammatory demyelinating polyneuropathy, Chronic relapsing multifocal osteomyelitis, Chronic lyme Disease (Chronic lyme Disease), Chronic obstructive pulmonary Disease, Churg-Strauss Syndrome, cicatricial pemphigoid, celiac Disease (Coeliac Disease), cochlear vestibular Syndrome, cold agglutinin Disease, Complement component2deficiency (complementary component2 deficiency), cerebral arteritis, CREST Syndrome, Crohn's Disease, Cushing's Syndrome, Cutaneous leukocytic vasculitis (Cutanous leukocytic angiitis), Dego's Disease, Degken's Disease, herpetiform dermatitis, dermatomyositis, type 1 diabetes, divergent Cutaneous systemic sclerosis (Diffusca systemic sclerosis), systemic lupus erythematosus Syndrome, Leptoderma lupus erythematosus, inflammatory bowel Disease, uterine lupus erythematosus, inflammatory bowel Disease, uterine lupus erythematosus, and herpes zoster's attachment, herpes zoster's Disease, herpes, Eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, idiopathic mixed cryoprecipital globulinemia (Essential mixed cryoglobulinemia), Evan's syndrome, progressive ossified fibrodysplasia, fibromyalgia/fibromyositis, and fibroalveolar blepharitisInflammation (fibrotic aviolitis), gastritis, Gastrointestinal pemphigoid (Gastrointestinal pemphigoid), giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-barre syndrome (Guillain-barre syndrome), Hashimoto's encephalitis (Hashimoto's encephalitis), Hashimoto's thyroiditis (Hashimoto's thyroiditis), hemolytic , allergic purpura (Henoch-schonelnpurpa), herpes gestationis, rheumatic hidradenitis, hysteritis syndrome (hughe syndrome), hypogammaglobulinemia, Idiopathic Inflammatory demyelinating disease (Inflammatory interstitial demyelatinizing disease), Idiopathic pulmonary fibrosis, thrombocytopenia, IBS, Inflammatory bowel disease (irritable bowel syndrome), IBS, juvenile myelopathy, IBS (Inflammatory bowel disease), juvenile myelopathy, IBS, Inflammatory bowel disease, IBS (Inflammatory bowel disease), juvenile myelopathy, Inflammatory bowel disease, IBS, juvenile myelopathy, Inflammatory bowel syndrome, IBS, juvenile myelopathy, Inflammatory bowel syndrome, Inflammatory bowel disease, IBS, Inflammatory bowel syndrome, IBS, kawasaki's Disease, Lambert-Eaton myasthenia syndrome (Lambert-Eaton muscular syndrome), Leucocytotic angiitis, lichen planus, lichen sclerosus, Linear IgA Disease, Lou Gehrig's Disease, Luo-Guick's Disease, Lupus lupus, lupus erythematosus, Magnetic syndrome (Majeed syndrome), Meniere's Disease, microscopic polyangiitis, Mi-Fisher syndrome (Miller-Fisher syndrome), mixed connective tissue Disease, scleroderma, Mucro-Haber syndrome (Murchase-Habermann Disease), Weldii syndrome (Muckle-Wells syndrome), multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcosis (Narcolepsy), neurooculitis, myoclonus ocularis-oculus syndrome (Opclonus-oculus syndrome), myoclonus-ichthyosis syndrome (Ophiomyxoma-iches syndrome), multiple sclerosis, myasthenia gravis, myofascicularis, narcosis (Narcolepsy), scleroma, myofasciosis, myofascicular syndrome, myofascicular Disease, myokeratosis, oddthyroiditis, recurrent rheumatism, PANDAS, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Trauner's syndrome, Parsonnage-Turnerstronme, Pars planitis, pimple, and herpesSores, pemphigus vulgaris, pernicious anemia, Perivenous encephalomyelitis (Pervenous encephalomyelitis), POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, Progressive inflammatory neuropathy (Progressive inflammatory neuropathy), psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red blood cell regeneration disorder, Ramusson encephalitis (Rasmussen ' sensory cephalis), Raynaud's phenomenon (Raynaud phenon), recurrent polychondritis, Rauter's syndrome, restless leg syndrome, retroperitoneal fibrosis, Rheumatoid arthritis, rheumatic fever (Rhumoheatofecter), sarcoidosis, schizophrenia, Schschschschschschschmitt syndrome (midschmitt syndrome), Niger syndrome, schlem's syndrome, scleroderma syndrome (Schlem's syndrome), sclerodermasyndrome), spondyloarthropathy, mucosis syndrome, Still's disease (Still's disease), stiff person syndrome, Subacute Bacterial Endocarditis (SBE), susacs syndrome (Susac ' ssyndrome), acute febrile neutrophilic skin disease (Sweet syndrome), sydenham's chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, toro-hunter syndrome (Tolosa-Hunt syndrome), transverse myelitis, ulcerative colitis, Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy (undivided sponalothropathology), vasculitis, baifeng, gewerner's granuloma (Wegener's granulomatosis), Wilson's syndrome (Wilson's syndrome) and sydow-Aldrich syndrome (wit syndrome).

In other preferred embodiments of aspects of the invention, the skin disease is selected from the group consisting of Acneiform eruptions (Acneiform eruptions), Autoinflammatory syndromes (Autoinflammatory syndromes), Chronic blistering (Chronic blistering), symptoms of mucous membranes (Conditions of the mucous membranes), symptoms of cutaneous appendages (Conditions of the skin aperpendants), symptoms of subcutaneous fat (Conditions of the subcutaneous lipid), congenital abnormalities, connective tissue diseases (such as abnormalities of dermal fibers and elastic tissue), dermal and subcutaneous growth, dermatitis (including atopic dermatitis, contact dermatitis, eczema, pustular dermatitis and seborrheic dermatitis), pigmentation disorders, Drug eruptions (Drug eruptions), endocrine-related skin, Eosinophilic skin diseases (eosinophicic skin), epidermal nevi, neoplasms (neoplasms), cystic dermatitis, erythrodermatitides, erythematoid skin infections, licheniform eruptions, lymphatic-related skin diseases, melanotic nevi and neoplasms (including melanoma), monocyte-related and macrophage-related skin diseases, mucinosis (Mucinoses), Neurocutaneous syndrome (neuro dermal), non-infectious immunodeficiency-related skin diseases, nutrition-related skin diseases, Papulosquamous hyperkeratosis (Papulosquamous hyperkeratotic) (including Palmoplantar keratoderma), pregnancy-related skin diseases, pruritus, psoriasis, Reactive neutrophilic skin diseases (Reactive neutrophilic), intractable Palmoplantar eruptions (Recalcitrant palmoplantarpretanoprotection), skin diseases caused by metabolic errors, skin diseases caused by physical factors (including ionizing radiation-induced skin diseases), urticaria and angioedema, vascular-related skin diseases.

In other preferred embodiments of each aspect of the invention, the endocrine disorder is selected from the group consisting of adrenal disorders, glucose metabolism disorders, thyroid disorders, calcium homeostasis disorders and metabolic bone disorders, pituitary gland disorders and sex hormone disorders.

In other preferred embodiments of each aspect of the invention, the ocular disease is selected from the group consisting of disorders of the eyelid, lacrimal system, and lacrimal passage from H00-H06, disorders of the conjunctiva from H10-H13, disorders of the sclera, cornea, iris, and ciliary body from H15-H22, disorders of the lens from H25-H28, disorders of the choroid and retina from H30-H36 (including H30 chorioretinitis, other disorders of the choroid from H31, disorders of the chorioretina from H32 classified elsewhere, detachment and fragmentation of the retina from H33, H34 retinal vessel occlusion, other retinal disorders from H35, and disorders of the retina from H36 classified elsewhere), disorders of the vitreous body and vitreous globe from H40-H42 glaucoma, disorders of the vitreous body and vitreous globe from H43-H45, disorders of the optic nerve and visual pathway from H46-H48, disorders of the extraocular muscles from H49-H52, disorders of the globe, Accommodation and refractive disorders, H53-H54.9 vision disorders and blindness, and other disorders of the H55-H59 eye and accessory organs.

In other preferred embodiments of aspects of the invention, the neurological disorder is selected from the group consisting of a dysesthesia, acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum dysplasia, agnosia, Aicardi syndrome (Aicardi syndrome), Alexanderdisease (Alexanderdisease), hemiparesis, paraphenosis, Alpers 'disease, cross-limb paralysis, alzheimer's disease, amyotrophic lateral sclerosis (see motor neuron disease), agathis, angels syndrome, angiomatosis, hypoxia, aphasia, arachnoid cyst, arachnoiditis, Arnold-chia malformation, arteriovenous malformations, ataxia-dysangiectasia, attention-deficit hyperactivity disorder, auditory processing disorder, autonomic dysfunction, backache, back pain disorder, Batten disease (Batten disease), Behcet ' S disease, Bell ' S palsy, benign idiopathic blepharospasm, benign intracranial hypertension, bilateral quota of multiple cerebella loops, Binswanger ' S disease, blepharospasm, Blacker-Sutzberg syndrome, brachial plexus nerve injury, brain abscess, brain injury, brain trauma, brain tumor, Brown-Secker syndrome, Canavan disease, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelinating disease, central nuclear myopathy, head disorder (Ceric disorder), brain aneurysm, cerebral artery diseaseSclerosis, brain atrophy, cerebral megalopathy, cerebral palsy, cerebral vasculitis, cervical stenosis, peroneal muscular atrophy, azimuthally disorder (Chiari malalformation), chorea, chronic fatigue syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic pain, Kohlerdi's syndrome (coffee Lowry syndrome), coma, complex area pain syndrome, compressive neuropathy, congenital bilateral facial paralysis, corticobasal degeneration, cerebral arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, giant cell inclusion body disease (Cytomelic infection bone disease) (Cidybd), cytomegalovirus infection, Dandan-Walker syndrome (Walker-Walker syndrome), Darsson disease (Darsoney disease), Klebsiella syndrome (Klebsiella palsy), and Dedydrome disease (Dedydrome), Deterner-Sottas disease (Dejerine-Sottas disease), delayed sleep phase syndrome, dementia, dermatomyositis, developmental movement disorder, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, familial autonomic dysfunction, computational difficulties, writing difficulties, dyslexia, dystonia, empty butterfly saddle syndrome, encephalitis, cerebral bulging, cerebral trigeminal neurovascular disease, fecal incontinence, epilepsy, Burserger's palsy (Erb's palsy), erythromelalgia, essential tremor, Fabry's disease, Fahr's syndrome, syncope, familial spastic paralysis, Febrile convulsions (Febrile sezars), Fichel's syndrome (Fisher syndrome), Friedreich's ataxia (Friedreich's syndrome), fibromyalgia, Gaucher's syndrome, Gothers syndrome (Geher's syndrome), Friedreich's ataxia (Friedreich's syndrome), Giant cell arteritis, giant cell inclusion body disease, globoid cell leukodystrophy, gray matter ectopy, Guillain-Barre syndrome, HTLV-1 related myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, polyneuritis type hereditary ataxia, ear herpes zoster, and Pingshan syndrome(Hirayama syndrome), forebrain anaclasis, Huntington's disease, hydrocephalus anencephalia, hydrocephalus, cortisol hyperplasia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, dyschromatosis, phytanic acid storage disease in infants, infant Raufson's disease (Infantile Refsum disease), Infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, Zhubert syndrome, Karak syndrome, Karens-Seker syndrome, Kearns-Sayre syndrome, Ganminger disease (Kennedy disease), Kinsbourne syndrome, Clinopril-Filler syndrome (Klippel-Feilsyndrome), Krebs disease (Krebs disease), Kuraker-Weldrain syndrome, and Larward-Verlag (Laurar-Laurar disease), Laurabernet disease (Laurar's disease), Lauraberber's disease (Lauraberware disease), Lauraber's disease (Lauraber's disease, Lauraber's syndrome, and Lauraber's disease (Lauraber's disease), Acquired aphasia associated with epilepsy (Landau-Kleffner syndrome), extramedullary (Walenburg) syndrome, learning disorders, Leigh's disease, Lento-Karschner syndrome, Lennox-Gastaut syndrome, Lesch-Nernel syndrome, leukodystrophy, Lewy body dementia, lissencephaly, atresia syndrome, Lougeri's disease (see motor neuron disease), lumbar disc herniation, lumbar spinal canal stenosis, Lyme disease (Lyme disease) -nervous system sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megabrain, Melkerson-Roth syndrome, Meltho-Joseph disease (Melknier syndrome), Melkerschenne meningitis, Merkel meningitis disease (Melkerschen disease), Merkshire syndrome, Melkerson syndrome, Merkinjure disease, Lennus syndrome, Lennox-Gastan-Gastaut syndrome, Leysonie-Lexu-Lexus syndrome, Lexus-Lexus syndrome, Leisha Leishi, Lexus syndrome, Leisha, metachromatic leukodystrophy, head teratocarcinosis, amebocia, migraine, Miller fishery syndrome (Miller Fishersyndrome), minor stroke (Mini-stroke) (transient ischemic attack), mitochondrial myopathy, morbikes syndrome, single limb muscular atrophy (monomer amyotrophy), motor neuron disease, motor skill disorder, smog disease, mucopolysaccharidosis, multiple cerebral infarctionDementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis (myalgic phalyositis), myasthenia gravis, diffuse sclerosis due to myelin destruction (myofascial difussclerosis), infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, myotonia congenita, Narcolepsy (Narcolepsy), neurofibromatosis, malignant neuroleptic syndrome, neurological manifestations of AIDS, neurological sequelae of lupus, neuromyotonia nervosa, neuronal ceroid lipofuscinosis, neuronal migration abnormalities, niemann-pick disease, non-24-hour sleep-arousal syndrome, non-learning disorder of the non-verbal type, oxaliplatin-maillard syndrome (Mco' Sullivan-Leod), neuralgia, dystrony syndrome, dystrophia spinal column neuroleptogenesis, macrobrachial syndrome (Ohtahia sytha), neuroleptic atrophy of the small bridge of brain, neuroleptic atrophy, Strabismus syndrome, optic neuritis, orthostatic hypotension, overuse syndrome, persistent afterimage, paresthesia, parkinson's disease, myotonia congenita, paraneoplastic disease (paraneoplastic diseases), Paroxysmal attacks (Paroxysmal attacks), parp-lodesh syndrome, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, persistent vegetative state, pervasive developmental disorder, optical sneeze reflex, phytanic acid disease, Pick's disease, nerve pinching, pituitary tumour, PMG, polio, polymaleic gyrus, polymyositis, cerebral puncture, polio late syndrome, Post Herpetic Neuralgia (PHN), postinfectious encephalomyelitis, postural hypotension, Prader-Willi syndrome (proder-Willi syndrome), primary lateral sclerosis, paresthesia, parkinson's disease, congenital myotonia, poliomyelitis, peripheral neuropathy, and cervical spondylosis, Prion diseases, progressive facial hemiatrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, pseudoencephaloma, rabies, lamuscle-hunter syndrome (Ramsay-Hunt syndrome) (types I and II), lamssons encephalitis (Rasmussen's encephalitis), reflex neurovascular dystrophy, Refsum disease, repetitive movement disorders, repetitive stress injuries, restless leg syndrome, retrovirus-associated spinal cord diseaseMyelopathy, Rett syndrome, Reiz's syndrome, rhythmic movement disorder, Romberg syndrome, chorea, Sandhoff disease, schizophrenia, diffuse periaxial encephalitis, schizoencephalus, dysesthesia, Septo-optic dyssplasia, shocked infant syndrome, herpes zoster, summer-Drder syndrome, Xiaoglen syndrome, and Zoogloea syndromesyndrome), sleep apnea, narcolepsy, satiety (snatation), Sotos syndrome (Sotos syndrome), spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, Steele-Richardson-Olszewski syndrome, stiff person syndrome, stroke, Sjogren's syndrome, subacute sclerosing panencephalitis, subcortical atherosclerotic encephalopathy, superficial iron deposition disease, Siden-Hamm's chorea, syncope, synaesthesia, syringomyelia, tarsal syndrome, tardive dyskinesia, sacral canal (Tarlov cyst), Tay-saxophone disease (Tay-Sachs disease), temporal arteritis, wind, tetanus, spinosyndrome syndrome, myotonic cataracts, trigeminal neuralgia (Tic) and trigeminal neuralgia (Tic), Todey paralysis (Todd's paralysis), Tourette's syndrome (Tourette syndrome), toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, Trigeminal neuralgia (Trigeminal neuralgia), tropical spastic paraplegia, trypanosomiasis, tuberous sclerosis, Von Hippel-Lindau disease, Williams encephalyitis (Viliuisk Encephaliomyelitis), Valenberg's syndrome (Walnberg ' ssdrome), Wallich-Hoffman syndrome (Werdnig-Hoffman disease), Westware syndrome, waving injury (Whiplash), Williams syndrome, Wilson's disease and Zhao-Zygler syndrome (Z-Gray syndrome)ellweger syndrome).

In other preferred embodiments of each aspect of the invention, the cardiovascular disease is selected from the group consisting of aneurysm, Angina (angiona), atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack), and peripheral vascular disease.

In other preferred embodiments of each aspect of the invention, the cardiovascular disease is not systemic lupus erythematosus.

In another aspect, the present invention provides a method of diagnosing a disease in a subject using a method of analyzing a blood sample according to the present invention. Thus, in another preferred embodiment of the method of the invention, the method of analyzing a blood sample according to the invention is part of a method of diagnosing a disease in a subject, and wherein the presence of the disease marker in the nucleic acid fraction of the anucleated blood cell extract is indicative of the subject suffering from the disease.

In another aspect, the present invention provides a method for determining the efficacy of a disease treatment in a subject, comprising the steps of:

at a first time point, analyzing a blood sample of a subject for the presence of a disease marker using a method of analyzing a blood sample according to the invention, thereby providing a first value as a level of the disease marker in the subject;

at a second time point (earlier or later than the first time point, preferably later than the first time point), analyzing the subject's blood sample for the presence of a disease marker using a method of analyzing a blood sample according to the invention, thereby providing a second value as the level of the disease marker in the subject, wherein the subject has undergone disease treatment between the first time point and the second time point; and

comparing the first value and the second value to determine the efficacy of the disease treatment in the subject.

The skilled person will appreciate that treatment prior to a first time point, and subsequent measurement at a second (later) time point between which no disease treatment occurred, are included in aspects of the invention for determining the efficacy of a disease treatment.

In another aspect, the invention provides a method for determining the stage of a disease. To determine the stage of the disease, it is advantageous to correlate the value of the disease marker determined by the method of the invention with the disease stage. The single measurement of the disease marker is then compared to one or more reference values to obtain an indication of the disease stage.

In another aspect, the present invention provides a method for determining the stage of a disease in a subject, comprising the steps of:

analyzing a blood sample of a subject for the presence of a disease marker using a method of analyzing a blood sample of a subject according to the present invention, thereby providing a test value as the level of the disease marker in the subject;

providing a reference value for the level of the disease marker, wherein the reference value is associated with a specific stage of disease; and

comparing the test value and the reference value to determine the stage of the disease in the subject.

In a further aspect, the present invention provides a kit of parts suitable for carrying out the method of the invention as described above, the kit comprising packaging material comprising at least one of:

a container for containing anucleated blood cells, preferably thrombocytes, separated from a blood sample;

a reagent for extracting nucleic acids from the anucleated blood cells;

reagents for selectively amplifying a disease-specific marker as described above, such as a disease-specific mutation in a gene of a nucleated cell of a subject or a disease-specific expression profile of a nucleic acid from a nucleated cell of the subject, from nucleic acid extracted from the anucleated blood cells, for example, by reverse transcriptase polymerase chain reaction amplification; and

printed or electronic instructions for carrying out the method of the invention as described above;

the kit further comprises:

a reference for the disease marker, wherein the reference indicates the presence or absence of the disease marker in the anucleated blood cell-extracted nucleic acid fraction.

In a preferred embodiment of the kit according to the invention, the reference is a reference value for the level of nucleic acid comprising the disease-specific mutation in anucleated blood cells of a healthy control subject or a control subject with a disease; or wherein the reference is, for example, a reference expression profile for a plurality of mrnas in anucleated blood cells from a healthy control subject or from a control subject having a disease.

In another preferred embodiment of the kit according to the invention, the reagents or instructions are selected from particles or fluorescent marker-labeled antibodies against anucleated blood cells (preferably fluorescent marker-labeled anti-thrombocytes antibodies), instructions for bead-based anucleated blood cell separation (preferably thrombocyte separation), instructions for FACS sorting of anucleated blood cells (preferably thrombocytes), instructions for recovery of anucleated blood cells (preferably thrombocytes) by centrifugation or instructions for negative selection of non-anucleated blood cell fractions (preferably non-thrombocyte fractions).

In a further aspect, the present invention provides a device for diagnosing a disease, the device comprising a support and at least one agent for specifically determining the level and/or activity of at least one nucleic acid mutant in a sample of anucleated blood cells of a subject, the agent being attached to the support; and a computer-readable medium having computer-executable instructions for implementing the method of the present invention as described above.

In a preferred embodiment of the device according to the invention, the at least one reagent is an oligonucleotide probe or a sequencing primer.

In a preferred embodiment of the device according to the present invention, the device comprises a lateral flow device, dipstick or cartridge for performing a nucleic acid hybridization reaction between the anucleated blood cell-extracted nucleic acid and the at least one nucleic acid mutation-specific amplified primer or oligonucleotide probe or between the anucleated blood cell-extracted nucleic acid and the plurality of gene-specific amplified primers or oligonucleotide probes for providing a disease-specific gene expression profile.

Drawings

FIG. 1: shown is the RNA expression profile analyzed using Agilent Bioanalyzer Picochip, where the length of the RNA (number of nucleotides) is on the X-axis and the amount of RNA (expressed in fluorescence units) is on the Y-axis. RNA from microvesicles in the serum fraction (1A), RNA from microvesicles in the plasma fraction (1B) or RNA from thrombocytes (1C) is described herein. The figures show that: 1) RNA is present in microvesicles in serum and plasma and in thrombocytes; 2) microvesicles isolated from a plasma sample contain less RNA than microvesicles isolated from a serum sample; and, 3) thrombocytes isolated from plasma samples contain RNA of various sizes, comprising a significant portion of relatively long RNA strands (> 200 nucleotides (nt), and even > 1000 nucleotides).

FIG. 2: results of a study showing tumor-derived genetic material found in thrombocytes from patients with brain tumors. Blood samples (whole blood vessels (serum (S)) and anticoagulated EDTA blood (plasma (P)) from patients (P1-P14) were taken from plasma tubes, thrombocytes (T) were collected by centrifugation protocol thrombocytes were collected from healthy individuals (C1-C6) as controls, some patients lacking serum samples are indicated by X in FIG. 2, and some patients with pooled serum and plasma samples are indicated with SP in figure 2, nested PCR was used for RNA detection, mutant EFGRvIII (V3) could be detected in 4 (27%) of 15 glioblastoma patients (P4, P5, P9, P10) thrombocytes, consistent with published literature, mutated EFGRvIII was found in 20% of high grade gliomas in the published literature (Liu et al 2005.) these experiments do provide evidence for the principle, i.e. the recognition of thrombocytes by tumor-derived nucleic acids, can be used as a source of biomarkers for the diagnosis of cancer.

FIG. 3: (A) u87 glioma-derived microvesicles were labeled with PKH67 green fluorescent dye, and the U87 glioma-derived microvesicles were incubated with isolated platelets. After incubation for 15min and 60min in the presence and absence of microvesicles, platelets were washed and FACS analysis of PKH67 fluorescence was performed. In addition, platelets were stained and analyzed by confocal microscopy to determine microbubble uptake. Following incubation with microvesicles under different conditions, RNA was isolated from RNase-treated platelets. RT-PCR was performed to detect RNA of EFGRvIII. MV/MVEFGRvIII: microvesicles isolated from U87/U87-EFGRvIII cells. (B) RNA was isolated from platelets from healthy control subjects or glioma patients and analyzed by RT-PCR. Corresponding glioma tissue biopsies were used as controls. PC = U87-EGFRvIII RNA; NC = H20; nd = undetermined; indicates a positive signal. (C) Performing gene expression array analysis on the RNA in (B). The left panel shows the heat map of the top 30 (top-30) glioma biomarkers. The right panel depicts the individual expression levels of the top 10 RNAs. Dotted line = BG (background).

FIG. 4: RNA was isolated from platelets from healthy control subjects (n = 8) and prostate cancer patients (n = 12) and RT-PCR analysis of PCA3, PSA and GAPDH was performed. Indicates weak positive.

FIG. 5: the probe sequences used to detect the genes shown in figure 3C are shown.

Detailed Description

As used herein, the term "cancer" refers to a disease or disorder resulting from the proliferation of oncogenically transformed cells. "cancer" shall encompass any one or more of a wide range of benign or malignant tumors, including those that are capable of growing, for example, via lymphatic and/or blood stream invasion and metastasizing across the human or animal body or portions thereof. Although the present invention is directed in particular to the diagnosis or detection of malignant tumors and solid cancers, as used herein, the term "tumor" encompasses both benign and malignant tumors or solid masses. The cancer further includes, but is not limited to, carcinomas, lymphomas or sarcomas, such as ovarian, colon, breast, pancreatic, lung, prostate, urinary tract, uterine, acute lymphocytic leukemia, Hodgkin's disease, small cell lung, melanoma, neuroblastoma, glioma (e.g., glioblastoma), and soft tissue sarcomas, lymphomas, melanomas, sarcomas and adenocarcinomas. In a preferred embodiment of each aspect of the invention, platelet cancer is disclaimed.

As used herein, the term "cancer-derived" refers to cells originating from a cancer or cancer.

The term "cancer-derived nucleic acid" is understood to mean any nucleic acid that is indicative of a cancer in a subject, in particular and in most embodiments, DNA or RNA that is indicative of a mutation of a mutant gene present in the cancer, which mutant gene is expressed by or present in a cancer cell of the subject, and whose nucleic acid sequence is altered relative to a normal gene of a healthy control subject. The term "cancer-derived nucleic acid" shall also comprise: (i) a nucleic acid that is produced, expressed or present in a cancer cell (but not in a normal healthy (non-cancer) cell), or whose production or expression is altered (increased or decreased) by or in a cancer cell relative to a normal cell; or (ii) a nucleic acid produced, expressed or present in a normal cell (but not by or in a cancer cell). Thus, the nucleic acid need not be a mutant nucleic acid having a mutant sequence, but can be a normal nucleic acid having a wild-type (non-cancer) sequence, but whose expression profile or level in a cancer cell is altered relative to a normal cell. In a preferred embodiment, the cancer-derived nucleic acid is a mutated nucleic acid (DNA, cDNA or RNA), preferably an RNA transcript, specific for cancer. In another highly preferred embodiment, the cancer-derived nucleic acid is a nucleic acid expression profile (as described in detail herein) that is indicative of being cancer-derived or cancer-specific.

As used herein, the term "cancer marker" refers specifically to a cancer marker gene or cancer marker gene expression profile. As used herein, the term "cancer marker gene" refers to a gene whose sequence or expression level (alone or in combination with other genes) is associated with cancer or cancer prognosis (prognosis). The correlation may involve an increase or decrease in expression of the gene, which is reflected in an increase or decrease in the presence of the RNA expression product of said gene in the nucleic acid portion obtainable from the thrombocytes. For example, expression of a gene may be indicative of cancer, or a loss of gene expression may be associated with poor prognosis in a cancer patient. In the case of prostate cancer AMACR, PCA3 and PSA are suitable cancer markers. In the case of colorectal cancer, KRAS mutations are suitable cancer markers. In the case of lung cancer, EGFR mutations are suitable cancer markers. In the case of melanoma, BRAF mutations are suitable cancer markers. In the case of gliomas, EGFRvIII mutations are suitable cancer markers. Other suitable cancer markers may be derived from tables 1 and 2 as provided herein, or from examples or figures. The skilled artisan will appreciate that many other cancer markers may be utilized in various aspects and embodiments of the present invention.

As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of cancer progression. Criteria for determining the stage of a cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body, and the location to which the cancer has spread (e.g., within the same organ or region of the body, or to another organ).

The term "cancer" in the terms "cancer derived", "cancer marker gene" and/or "stage of cancer" may be generalized to the term "disease" (as defined for cancer), as the definition for cancer may generally apply to all diseases described herein.

As used herein, the term "disease-derived" refers to cells derived from a disease or disease.

The term "disease-derived nucleic acid" is understood to mean any nucleic acid that is indicative of a disease in a subject, in particular and in most embodiments, DNA or RNA that is indicative of a mutation of a mutant gene present in a disease, which mutant gene is expressed by or present in a diseased cell of a subject, and whose nucleic acid sequence is altered relative to a normal gene of a healthy control subject. The term "disease-derived nucleic acid" is understood to encompass: (i) a nucleic acid that is produced, expressed or present in a diseased cell (but not in a normal healthy (non-diseased) cell), or whose production or expression is altered (increased or decreased) by a diseased cell or in a diseased cell relative to a normal cell; or (ii) a nucleic acid produced, expressed or present in a normal cell (but not in a diseased cell or in a diseased cell). Thus, the nucleic acid need not be a mutant nucleic acid having a mutant sequence, but can be a normal nucleic acid having a wild-type (non-disease) sequence, but whose expression profile or level in diseased cells is altered relative to normal cells. In a preferred embodiment, the disease-derived nucleic acid is a mutated nucleic acid (DNA, cDNA or RNA), preferably an RNA transcript, specific for the disease. In another highly preferred embodiment, the disease-derived nucleic acid is a nucleic acid expression profile (as described in detail herein) indicative of disease origin or disease specificity. In a preferred embodiment, the disease-derived nucleic acid does not comprise a cancer-derived nucleic acid. In yet another preferred embodiment, the disease-derived nucleic acid does not comprise vascular disease-derived nucleic acid, and/or systemic lupus erythematosus-derived nucleic acid. In a preferred embodiment, the disease-derived nucleic acid does not comprise sickle cell disease-derived nucleic acid. In a preferred embodiment, the disease-derived nucleic acid does not comprise Alzheimer's disease-derived nucleic acid. In a preferred embodiment of the invention and embodiments thereof, the disease-derived nucleic acid does not comprise a CD109 nucleic acid. In a preferred embodiment of the invention and embodiments thereof, the disease-derived nucleic acid does not comprise a megakaryocyte-derived nucleic acid. In a preferred embodiment of the invention and embodiments thereof, the disease-derived nucleic acid does not include nucleic acids derived from a disease associated with pathological megakaryocyte and/or platelet function.

As used herein, the term "disease marker" refers specifically to a disease marker gene or disease marker gene expression profile. As used herein, the term "disease marker gene" refers to a gene whose sequence or expression level (alone or in combination with other genes) is correlated with a disease or disease prognosis. The correlation may involve an increase or decrease in expression of the gene, which is reflected in an increase or decrease in the presence of the RNA expression product of said gene in the nucleic acid portion obtainable from the thrombocytes. For example, expression of a gene may be indicative of a disease, or a loss of gene expression may be associated with a poor prognosis in a patient with a disease. In a preferred embodiment, the disease marker gene is not the CD109 gene.

As used herein, the term "stage of a disease" refers to a qualitative or quantitative assessment of the level of disease progression. Criteria for determining the stage of a disease include, but are not limited to, whether the disease has spread to other parts of the body, and the location to which the disease has spread (e.g., within the same organ or region of the body, or to another organ).

As used herein, the term "disease" may refer to cancer, autoimmune diseases, skin diseases, eye diseases, endocrine diseases, neurological disorders, and cardiovascular diseases.

As used herein, the term "disease" may refer to autoimmune diseases, skin diseases, eye diseases, endocrine diseases, neurological disorders, and/or cardiovascular diseases.

As used herein, the term "disease" may refer to autoimmune diseases, skin diseases, eye diseases, endocrine diseases, and/or neurological disorders.

As used herein, in some preferred embodiments, the term "disease" may not refer to: cancer, cardiovascular disease, systemic lupus erythematosus, sickle cell disease, Alzheimer's disease, disease associated with pathological platelet function, and/or disease associated with pathological megakaryocyte function.

Thus, the means and methods of the invention may be used to detect diseases other than or not cancer, such diseases including, for example, the following autoimmune diseases: achlorhydria autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, anti-synthetase syndrome, polyarthritis, atopy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral nerve disease, autoimmune pancreatitis, autoimmune polyendocrine gland syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, inflammatory bowel disease, inflammatory, Barlow's/Barlow's concentric sclerosis, Behcet's syndrome, Berger's disease, Bischner's encephalitis, Blu's syndrome, bullous pemphigoid, Cashmere's disease, celiac disease, Chagas' disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, celiac vestibular disease, cold agglutinin disease, complement component2deficiency, craniotomitis, CREST syndrome, Crohn's disease, Cushing syndrome, cutaneous leukoclastic vasculitis, Dego's disease, Deleken's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, divergent cutaneous systemic sclerosis, Descemer's syndrome, discoid lupus erythematosus, eczema, endometriosis, lupus erythematosus, endometriosis, cervical spondyloschistosomiasis, cervical spondylopathy, and other diseases, Aconitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, acquired epidermolysis bullosa, erythema nodosum, idiopathic mixed cryoprecipital globulinemia, Evan's syndrome, progressive fibrodysplasia ossificans, fibromyalgia/fibromyositis, fibrositis alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves ' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Geben's thyroiditis, hemolytic blood, allergic purpura, herpes gestationis, hidradenitis suppurativa, Houseus syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, cystitis, inflammatory interstitial cystitis, inflammatory bowel disease, inflammatory bowel syndrome, Irritable Bowel Syndrome (IBS), juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Ilton myasthenia syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease, Luugreek disease, lupus erythematosus, Magidd syndrome, Meniere's disease, microscopic polyangiitis, Mi-Fisher syndrome, mixed connective tissue disease, scleroderma, Mu-Har's disease, Weldii syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromuscular neuritis, ocular cicatricial pemphigoid, ocular clonus-myoclonus syndrome, Aldthyroiditis, recurrent rheumatism, PANDAS, paracerebral degeneration, neoplastic sleeping proteinuria, Paul-Rodi syndrome, Pakis-Roodpasture's syndrome, Paget's-Erythroseus syndrome, Graetson-Ile syndrome, Graetson-Barre syndrome, Muetschnei-Barre syndrome, Murraya-Sphaeroken syndrome, Murra, Terna's syndrome, pars plana, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell regeneration disorder, Lamessen encephalitis, Raynaud's phenomenon, relapsing polychondritis, Laplace's disease, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnier's syndrome, scleritis, scleroderma, Shogren's syndrome, spondyloarthropathy, mucinous syndrome, Steyr's disease, stiff person's syndrome, Subacute Bacterial Endocarditis (SBE), Susaki syndrome, Sunsal's syndrome, Skoki syndrome, Graves's disease, Graves's syndrome, Graves' syndrome, Acute febrile neutrophilic dermatosis, sydner's chorea, sympathetic ophthalmia, takayasu's arteritis, temporal arteritis, Toloxa-Hunter syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome and Wiscott-Aldrich syndrome.

In addition to the above-mentioned diseases, aspects of the present invention can also be applied to prognosis and diagnosis of the following skin diseases: acne-like rash, auto-inflammatory syndrome, chronic blistering, symptoms of mucous membranes, symptoms of skin appendages, symptoms of subcutaneous fat, congenital anomalies, connective tissue diseases (such as anomalies of dermal fibers and elastic tissues), dermal and subcutaneous growth, dermatitis (including atopic dermatitis, contact dermatitis, eczema, pustular dermatitis and seborrheic dermatitis), pigmentation disorders, drug eruptions, endocrine-related skin, eosinophilic dermatoses, epidermal nevi, neoplasms, cysts, erythema, hereditary dermatoses, infection-related dermatoses, licheniform eruptions, lymph-related dermatoses, melanomas and neoplasms (including melanoma), monocyte-related dermatoses and macrophage-related dermatoses, mucinosis, neurocutaneous syndrome, non-infectious immunodeficiency-related dermatoses, nutrition-related dermatoses, papular squamous hyperkeratosis (including palmoplantar keratosis), pregnancy-related skin diseases, pruritus, psoriasis, reactive neutrophilic skin diseases, intractable palmoplantar eruptions, skin diseases caused by metabolic errors, skin diseases caused by physical factors (including ionizing radiation-induced skin diseases), urticaria and angioedema, vascular-related skin diseases.

In addition to the above-mentioned diseases, aspects of the present invention can also be applied to prognosis and diagnosis of the following endocrine diseases: adrenal disorders, glucose metabolism disorders, thyroid disorders, calcium homeostasis disorders and metabolic bone diseases, pituitary gland disorders, and sex hormone disorders.

In addition to the above diseases, aspects of the present invention may also be applied to prognosis and diagnosis of the following eye diseases: H00-H06 disorders of the eyelid, lacrimal system and lacrimal passage, H10-H13 disorders of the conjunctiva, H15-H22 sclera, cornea, iris and ciliary body disorders, H25-H28 lens disorders, H30-H36 choroidal and retinal disorders (including H30 chorioretinitis, other disorders of H31 choroid, chorioretinal disorders of H32 in diseases classified elsewhere, H33 retinal detachment and fragmentation, H34 retinal vascular occlusion, other retinal disorders of H35, and retinal disorders of H36 in diseases classified elsewhere), H40-H42 glaucoma, H43-H45 vitreous and vitreous disorders, H46-H48 optic nerve and visual pathway disorders, H49-H52 extraocular muscles, movements, accommodation, and refraction disorders of both eyes, H53-H54.9 vision disorders and blindness, and other disorders of H55-H59 eyes and accessory organs.

In addition to the above-mentioned diseases, aspects of the present invention can also be applied to prognosis and diagnosis of the following neurological disorders: dystonia, acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum dysplasia, agnosia, aicardia syndrome, alexander's disease, anorthrosis, paresthesia, alper's disease, cross-limb paralysis, alzheimer's disease, amyotrophic lateral sclerosis (see motor neuron disease), anencephaly, angelicae syndrome, hemangiomatosis, hypoxia, aphasia, apraxia, arachnoid cyst, arachnoiditis, arnoder-kiri malformations, arteriovenous malformations, ataxia telangiectasia, attention deficit hyperactivity disorder, auditory processing disorder, autonomic dysfunction, back pain, batten disease, behcet's disease, bell's palsy, benign idiopathic blepharospasm, benign intracranial hypertension, bilateral quota-determining cerebellar gyrus, binge's disease, blepharospasma, Brookfield-Sutzberg syndrome, brachial plexus injury, brain abscess, brain injury, brain trauma, brain tumor, Brown-Seaker syndrome, Canavan's disease, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelination, centronuclear myopathy, head disorders, cerebral aneurysms, cerebral arteriosclerosis, brain atrophy, cerebral gigantism, cerebral palsy, cerebrovascular disease, cervical stenosis, musculoskeletal atrophy, Alzheimer's disease, chorea, chronic fatigue syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic pain, Kohler's syndrome, coma, complex regional pain syndrome, compressed neuropathy, hirsutism, corticobasal degeneration, cerebral arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, Crohn's disease, Graves's disease, cumulative trauma disorder, Cushing's syndrome, Crohn's disease, cytomegalo Inclusion Body Disease (CIBD), cytomegalo virus infection, dandy-wack syndrome, a dyson disease, delmosil syndrome, crohn's palsy, derris-sottass disease, delayed sleep phase syndrome, dementia, dermatomyositis, developmental exercise disorder, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, familial autonomic dysfunction, computational difficulty, writing difficulty, dyslexia, dystonia, empty sphenoid saddle syndrome, encephalitis, cerebral bulging, cerebral trigeminal neurovascular tumor disease, fecal incontinence, epilepsy, bob's palsy, erythromelalgia, essential tremor, fabry disease, french syndrome, fainting, familial spastic paralysis, convulsions, fexil syndrome, friedrich's ataxia, fibromyalgia, gaucher disease, gerstman syndrome, giant cell arteritis, giant cell paralysis, and combinations thereof, Giant cell inclusion body disease, globuloleukodystrophy, gray matter ectopy, Guillain-Barre syndrome, HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, polyneuritis-type hereditary ataxia, herpes zoster, Pingshan syndrome, forebrain anaphalosis, Huntington's disease, hydrocephalus, cortisol hyperplasia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, pigment disorders, infantile phytanic acid disease, infantile Ruiffson's disease, infantile spasm, inflammatory myopathy, intracranial cysts, increased intracranial pressure, Verbert syndrome, Carrak syndrome, Carnsser-Selle syndrome, Gannedy's disease, Kisburner's syndrome, Clipel-Verier syndrome, inflammatory myopathy, intracranial pressure-increasing syndrome, intracranial pressure, Tourette's syndrome, cerebral paler's syndrome, cerebral paletto syndrome, cerebral paleness syndrome, and cerebral paler syndrome, Krabbe's disease, kurgebe-welan's disease, kuru, lafura's disease, lambert-eaton myasthenia syndrome, acquired aphasia with epilepsy, lateral bulbar (valency) syndrome, learning disorders, leigh's disease, lunge-gaster's syndrome, lesch-neen syndrome, leukodystrophy, lewy body dementia, cerebellar malformation, atresia syndrome, lou gehrick's disease (see motor neuron disease), lumbar disc herniation, lumbar spinal stenosis, lyme disease-nervous system sequelae, machado-joseph disease (spinocerebellar ataxia type 3), megabrain, visual manifestations, megabrain, meniere-roche syndrome, meniere's disease, meningitis, menkes disease, metachromatic leukodystrophy, small cephalic, migraine, visceroid-mediated dystrophia, neuro-mediated disorders, and other disorders, Mifare syndrome, stroke (transient ischemic attack), mitochondrial myopathy, mobius syndrome, single limb muscular atrophy, motor neuron disease, motor skills impairment, smog disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinating diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, tubular myopathy, congenital myotonia, narcolepsy, neurofibromatosis, malignant neuroleptic syndrome, neurological manifestations of AIDS, neurological sequelae of lupus, neuropathic myotonia, neuronal ceroid lipofuscinosis, neuronal migration abnormalities, niemann-pick disease, non-24-hour sleep-wake syndrome, nonverbal learning disorders, cognitive disorders, and, Oxalin-mclaud syndrome, occipital neuralgia, recessive spinal canal insufficiency syndrome, Protopanagen syndrome, olivopontocerebellar atrophy, strabismus syndrome, optic neuritis, orthostatic hypotension, overuse syndrome, persistent afterimages, paresthesia, Parkinson's disease, myotonia congenita, paraneoplastic disease, paroxysmal attacks, Paris-Ross syndrome, Pelizaeus-Merzbach disease, periodic paralysis, peripheral neuropathy, persistent vegetative state, pervasive developmental disorder, optical sneeze reflex, phytanic acid storage disease, pick's disease, nerve pinching, pituitary tumor, PMG, polio, polymyositis, cerebral puncture, postpolio syndrome, postherpetic neuralgia (PHN), postherpetic encephalomyelitis, hypotensive encephalomyelitis, hypotension, postherpetic neuralgia, Paradoxa (PHN), postherpetic encephalomyelitis, paradoxus syndrome, paradoxical neuralgia, spasticity, prader-willi syndrome, primary lateral sclerosis, prion diseases, progressive facial hemiatrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, pseudoencephaloma, rabies, lamarchia-hunter syndrome (type I and type II), lasmessen encephalitis, neurovascular dystrophy, refsum disease, repetitive movement disorders, repetitive stress injury, restless leg syndrome, retrovirus-associated myelopathy, leigh syndrome, raychia syndrome, rhythmic movement disorders, lobelia syndrome, chorea, sandhoff disease, schizophrenia, diffuse periaxial encephalitis, split brain, sensory dysesthesia, dystrophia, shock-induced infant syndrome, bandicoverruca, chard-de syndrome, schoglabran syndrome, sleep apnea, narcolepsy, satiation, depression, sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, Stery-Older-Karschner syndrome, stiff person syndrome, stroke, Sjorwort syndrome, subacute sclerosing panencephalitis, subcortical atherosclerotic encephalopathy, superficial iron deposition disease, Xidenham's chorea, syncope, synaesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, sacral cyst, Thai-saxophone disease, temporal arteritis, tetanus, spinotethered syndrome, myotonic cataract, thoracic outlet syndrome, trigeminal neuralgia, Todarby paralysis, Tourette syndrome, toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraplegia, spastic paraplegia, Trypanosomiasis, tuberous cerebral sclerosis, von willebrand's disease, willebrand encephalomyelitis, warenberg syndrome, warnik-hofmann disease, west syndrome, whip injury, williams syndrome, wilson's disease and zhao-weiwei syndrome.

In addition to the above-mentioned diseases, aspects of the present invention may also be applied to the prognosis and diagnosis of the following cardiovascular diseases: aneurysms, angina, atherosclerosis, cerebrovascular accidents (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack), and peripheral vascular disease. In preferred embodiments of the methods of the invention and embodiments thereof, as well as preferred embodiments of other aspects of the invention, the disease or cardiovascular disease is not systemic lupus erythematosus.

In a preferred embodiment of the method of the invention and its embodiments, as well as in preferred embodiments of other aspects of the invention, the disease is not a disease selected from the group comprising cancer, cardiovascular disease, systemic lupus erythematosus, sickle cell disease, alzheimer's disease, a disease associated with pathological platelet function, and/or a disease associated with pathological megakaryocyte function.

As used herein, "nucleic acid" comprises deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, the nucleic acid includes known analogs having the basic properties of natural nucleotides, as these analogs hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (peptide nucleic acids).

The term "RNA" refers to ribonucleic acids, RNA molecules that encode a protein product or do not encode a protein product (e.g., miRNA, but not other non-coding RNAs). RNA is transcribed from the DNA template.

As used herein, the term "mutant" refers to a nucleic acid compound, protein, molecule, vector, or cell that results from a mutation in a native wild-type coding sequence or subunit thereof (subbunit).

As used herein, the term "mutation" refers to any change in a native coding sequence by substitution (displacement), addition, deletion, insertion, cross-linking or other disruption, or by substitution of one or more nucleotides in the native coding sequence (including naturally occurring splice variants). The mutation particularly provides a gene that causes the cell to become a cancer cell. Such mutations include genetic, and acquired mutations of tumor suppressor genes and/or oncogenes.

By "amplifying" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to a nucleic acid sequence using at least one nucleic acid sequence as a template. Amplification systems include Polymerase Chain Reaction (PCR) systems, Ligase Chain Reaction (LCR) systems, nucleic acid sequence-dependent amplification (NASBA, Cangene, Missisoga, Ontario), Q-Beta replicase systems, transcription-dependent amplification systems (TAS), and Strand Displacement Amplification (SDA). See, e.g., Diagnostic Molecular microbiology. principles and applications, d.h. lasting, et al, editors, american society for microbiology, washington, d.1993. The amplification product is called an amplicon.

The term "hybrid" refers to a double-stranded nucleic acid molecule or duplex (duplex) formed by hydrogen bonding between complementary nucleotides. The term "hybridization" or "annealing" refers to the process by which a single strand of a nucleic acid sequence forms a duplex segment through hydrogen bonding between complementary nucleotides.

The term "oligonucleotide" refers to a short chain of nucleotide monomers (typically 6 to 100 nucleotides) linked by phosphorus-containing linkers (e.g., phosphodiesters, alkyl and aryl phosphates, phosphosulfuryl) or non-phosphorus-containing linkers (e.g., peptides, sulfamates, and others). Oligonucleotides may comprise modified nucleotides having modified bases (e.g., 5-methylcytosine) and modified sugar groups (e.g., 2-O '-methylribosyl, 2-O' -methoxyethylribosyl, 2 '-fluororibosyl, 2' -aminoribosyl, and the like). Oligonucleotides may be naturally occurring or synthetic molecules having circular, branched or linear shapes of double-and single-stranded DNA and double-or single-stranded RNA, and the DNA and RNA optionally contain domains capable of forming stable secondary structures (e.g., stem-loop structures and loop-stem-loop structures).

As used herein, the term "primer" refers to an oligonucleotide as follows: capable of annealing to amplification purposes thereby allowing DNA polymerase binding, serves as a point of initiation of DNA synthesis when placed under conditions in which primer extension products (complementary to the induced nucleic acid strands) are synthesized, i.e., in the presence of nucleotides and reagents for polymerization (such as DNA polymerase) and at an appropriate temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to direct the synthesis of the extension product in the presence of the reagents used for polymerization. The exact length of the primer will depend on many factors, including temperature and the source of the primer. As used herein, "bidirectional primer pair" refers to one forward primer and one reverse primer commonly used in the field of DNA amplification, such as in PCR amplification.

The term "probe" refers to a single-stranded oligonucleotide sequence capable of recognizing a complementary sequence in a nucleic acid sequence analyte of interest or a cDNA derivative thereof and forming a hydrogen-bonded duplex therewith.

Term(s) for"stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect the stability of a hybrid, e.g., temperature, salt concentration, pH, formamide concentration, and the like. These conditions can be empirically optimized to maximize specific binding of a primer or probe to its nucleic acid sequence of interest, but not to minimize specific binding. The terms used include the following conditions: under such conditions, the probe or primer will hybridize to its sequence of interest to a higher detectable degree than the other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and should be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be the thermal melting point (T) at a defined ionic strength and pH for a particular sequence_m) About 5 deg.c lower. T is_mThe following temperatures were used: approximately 50% of the complementary sequence of interest hybridizes (at a defined ionic strength and pH) to a perfectly matched probe or primer at that temperature. Typically, stringent conditions will be as follows: under these conditions, the salt concentration is less than about 1.0M Na at a pH of 7.0 to 8.3⁺Ions, typically about 0.01M to 1.0M Na⁺Ion concentration (or other salts); and a temperature of at least about 30 ℃ for short probes or primers (e.g., 10-50 nucleotides) and at least about 60 ℃ for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions or "reduced stringency conditions" include: hybridization was performed using a buffer solution of 30% formamide, 1M NaCl, 1% SDS at 37 ℃ and washing in2 XSSC at 40 ℃. Exemplary high stringency conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 ℃, and washes in 0.1 x SSC at 60 ℃. Hybridization procedures are well known in the art and are described, for example, in Current Protocols in molecular biology (John Wiley) by Ausubel et al&Sons inc., 1994).

As used herein, a "subject" includes (but is not limited to) a mammal, including: human, non-human primate, mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, gerbil, horse, monkey, sheep, or other non-human mammal; and, non-mammalian animals, including, for example, non-mammalian vertebrates (e.g., birds (e.g., chickens or ducks) or fish) and invertebrates. The subject may be a healthy animal or human subject undergoing a routine physical examination. Alternatively, the subject may be a subject at risk of a disease, e.g., a genetically predisposed subject, a subject with a medical and/or family history of cancer, a subject who has suffered carcinogens, occupational injury, environmental injury, and/or a subject who shows suspicious clinical signs of a disease (e.g., bloody or dark stool in stool, unclear pain, sweating, unclear fever, unclear weight loss to loss of appetite, changes in stool habits (constipation and/or diarrhea), tenesmus (feelings of incomplete bowel movement), anemia, and/or general weakness). According to another embodiment, the subject may be a patient diagnosed with a disease and undergoing routine physical examination, intermediate treatment.

As used herein, the term "thrombocyte" refers to a platelet, i.e., a small irregularly shaped cellular fragment that does not have a DNA-containing nucleus and that circulates in mammalian blood. The thrombocytes have a diameter of 2 to 3 μm and are derived from the disruption of precursor megakaryocytes. Platelets or thrombocytes lack nuclear DNA, but they retain some megakaryocyte-derived mRNA as part of their orthotopic origin. The average life span of thrombocytes is 5 to 9 days. Thrombocytes participate in and play a key role in hemostasis, leading to the formation of blood clots. In a preferred embodiment of the invention and its embodiments, the nucleic acid fraction extracted from the enucleated blood cells is not megakaryocyte-derived nucleic acid or megakaryocyte-derived RNA.

As used herein, the term "blood" refers to whole blood (including plasma and cells), and includes arterial blood, capillary blood, and venous blood.

As used herein, the term "nucleated cell" preferably refers to a Pasteur Gland cell (Bartholin's Gland cell), salivary Gland mucous cell, salivary Gland serous cell, Ebunner Gland cell (Von Ebner's Gland cell), mammary Gland cell, lacrimal Gland cell, cerumen Gland cell, excretory sweat Gland cell, apocrine Gland cell, Morel Gland (Glandof Gland cell), sebaceous Gland cell, Bowman's Gland, duodenal Gland (Brunner's Gland), seminiferous vesicle cell, prostate cell, bulbourethral Gland cell, Littley Gland (Gland of Littre cell), endometrial cell, isolated goblet cell, Gastric mucosa (Stomachiing mucous cell), Gastric zymogen (Gastric Gland zymogenic cell), Gastric Gland secretory cell, pancreatic acinar cell, Pangolian cell, pituitary II cell, anterior pituitary cell, etc, Large Cell neurosecretory cells, thyroid cells, parathyroid cells, adrenal cortical cells, Leydig cells, follicular intima cells, luteal cells, pericyclic cells, compact plaque cells, peripolar cells, mesangial cells, vascular and lymphatic endothelial fenestrated cells, vascular and lymphatic endothelial continuous cells, vascular and lymphatic endothelial splenocytes, synovial cells, serosal cells, squamous cells, columnar cells, dark cells, vestibular membrane cells, angiobasal cells, angiostriatal limbic cells, claudi us cells (Cell of Claudius), perifibrate cells (of Boettcher), choroid plexus cells, pia-arachnoid cells, Pigmented ciliary epithelial cells (Pigmented ciliary epithelial cells), non-Pigmented ciliary epithelial cells (non-Pigmented ciliary epithelial cells), corneal endothelial cells, and endothelial cells, Cells that are tethered (Peg cells), respiratory Ciliated cells, oviduct Ciliated cells, endometrial Ciliated cells, testicular Ciliated cells, Ciliated cells of the seminiferous tubules, Ciliated ependymal cells, epidermal keratinocytes, epidermal basal cells, keratinocytes, nail bed basal cells, medullary hair axis cells, cortical hair axis cells, hair axis cells of the epidermis, epidermal hair root sheath cells, External hair root sheath cells (External hair follicle cells), hair matrix cells, surface epithelial cells, stromal cells, urinary tract epithelial cells, auditory inner ear hair cells, auditory outer hair cells, primary sensory neurons, merkel cells, olfactory receptor neurons, photoreceptor cells, carotid body cells (blood pH sensors), hair cells, taste bud cells, cholinergic nerve cells, adrenergic nerve cells, peptidergic nerve cells, neuro, Inner pillar cells, outer pillar cells, inner finger cells, outer finger cells, limbal cells, hansen cells, vestibular organ supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, intestinal glial cells, astrocytes, neuronal cells, oligodendrocytes, spindle neurons, anterior lens epithelial cells, lens fibrillar cells containing crystallin, hepatocytes, adipocytes, hepatic storage adipocytes, glomerular parietal cells, glomerular podocytes, renal proximal tubule brush border cells (Kidney proximal tubule brush border cells), henry jacket (Loop of henle) thin segment cells, renal distal tubular cells, renal collecting duct cells, lung cells, pancreatic duct cells, non-striated duct cells, intestinal brush border cells, exocrine gland striated duct cells, gallbladder epithelial cells, and Kidney cells, Seminiferous tubule non-ciliated cells, epididymal host cells, epididymal basal cells, amelogenic epithelial cells, semilunar epithelial cells, ldistian interdental epithelial cells (Organ of Corti interdentate epithelial cells), loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, fibroblasts of other non-epithelial cell types, pericytes, nucleus pulposus cells, odontoblasts/cementoblasts, odontoblasts/dentin cells, hyaline chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts/osteocytes, osteoprogenits, vitreous cells, stellate cells, hepatic stellate cells, pancreatic stellate cells, skeletal muscle cells, satellite cells, cardiac muscle cells, smooth muscle cells, muscle epithelial cells, monocytes, macrophages of connective tissue, epithelial Langerhans cells, human embryonic stem cells, osteoclasts, dendritic cells, microglia, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, melanocytes, retinal pigment epithelial cells, oogonia/oocytes, sperm cells, spermatocytes, spermatogonia, sperm, ovarian follicular cells, sertoli cells (sertoli cells), thymic epithelial cells, and renal stromal cells.

Targeted therapy and personalized medicine are heavily dependent on the development of a spectrum of diseases (profiling) and concomitant diagnostics. Mutants of disease-derived nucleic acids can be highly predictive of response to targeted therapy. However, there is still a significant obstacle to the development of easily available high quality nucleic acids. Blood typically contains 150,000-350,000 thrombocytes per microliter, providing a highly available source of biomarkers for research and clinical applications. Furthermore, thrombocyte isolation is relatively simple and is a standard procedure in blood banks/hematology laboratories. Since platelets do not contain nuclei, the RNA transcripts required for their functional maintenance are megakaryocytes derived from bone marrow during the process of thrombocyte origin. It has now been found that thrombocytes can obtain RNA and/or DNA from cells other than megakaryocytes in a circulatory process via different delivery mechanisms. For example, tumor cells release large amounts of genetic material, some of which are secreted by microvesicles in the form of mutant RNAs. Thrombocytes, during their circulation in the bloodstream, take up the genetic material secreted by cancer cells and other diseased cells, acting as an attractive platform for the concomitant diagnosis of cancer and other diseases as indicated above (e.g. in the case of personalized medicine).

In the examples below, platelets isolated from healthy human control subjects are shown to have the ability to take RNA from RNA-containing microvesicles derived from human brain tumor cells (gliomas), these platelets then containing tumor-associated RNA, including for example mutated EGFRvIII mRNA in the case of glioma patients. Thus, circulating platelets isolated from glioma patients were determined to contain RNA biomarkers. RT-PCR was used to confirm that the mutant egfrviii mrna found in thrombocytes reflects the presence of gliomas.

The presence of tumor and/or disease-marker information is not specific to platelets from glioma patients, but is more generally applicable to a wide range of diseases as described herein. Messenger RNAs encoding the prostate cancer markers PCA3 and PSA could be demonstrated to be present in platelets from prostate cancer patients, whereas these markers were not present in platelets from healthy control subjects.

In addition to detecting genetic mutations associated with cancer or other diseases, the present inventors have also discovered that gene expression arrays can be used to classify thrombocyte nucleic acid samples as thrombocyte nucleic acid samples of subjects having a particular type of (solid tumor) cancer or other disease. It was confirmed that mRNA expression profiles obtained using nucleic acids extracted from platelets isolated from healthy control subjects or from glioma patients were significantly different. Distinct mRNA expression profiles were obtained and the smallest glioma biomarker signature could be detected, as shown in fig. 3C for the top 30 hits (top-30 hit). As shown in fig. 3C, the distinctive expression profile includes significant elevations in the expression of the following genes: WFDC1, Kremen1, DEF4A, ARG1, FKBP5, ACRC, ENST0328043, a _32_ P167111, MAP2, ECTL8, UNC13B, TP53I3, FDXR, BX119718, SORT1, PFN4, C1QTNF5, a _24_ P237896, PGLYRP1, SEC14L2, BC018626, MAOB, TCN1, AMOTL1, TSP50, a _24_ P927015, THC2325987, C18orf1, and LIN28 (some of these gene names refer to reference microarray record numbers such as oligonucleotide probes of Agilent Chip). It will be appreciated that this expression profile does not limit the scope of the invention, as the skilled person knows how to use the method of the invention to obtain other suitable gene expression profiles for other cancers or for other diseases in general.

The present inventors have now found that platelets contain cancer markers and disease markers in the form of tumor-derived or tumor-associated or disease-derived nucleic acids or nucleic acid expression profiles, and that these platelets can be used as diagnostic platforms for molecular expression profiles of cancer and other diseases as defined herein. This is very useful in the case of personalized medicine.

The present invention provides novel and easy to use methods to isolate circulating disease-derived material (e.g., disease markers as used herein) for genetic analysis. The present inventors isolated tumor-derived RNA from circulating thrombocytes, producing pure RNA, thereby providing a simple way to extract high quality RNA from small amounts of blood. Isolation and subsequent analysis of thrombocyte Nucleic Acid (NA) showed a significant increase in diagnostic sensitivity to circulating NA in blood.

The present inventors have found that circulating thrombocytes contain significant amounts of disease-derived RNA and/or DNA in disease patients. The disease-derived RNA and/or DNA displays unique genetic information about the disease that can be used to determine the type of disease, the extent of the disease, and possibly the susceptibility of the disease to treatment. In a preferred embodiment of the method or embodiment of the invention, the disease is not cancer.

In a preferred embodiment of the method or embodiment of the invention, the disease is not a vascular disease.

In a preferred embodiment of the method or embodiment of the invention, the disease is not systemic lupus erythematosus.

In a preferred embodiment of the method or embodiment of the invention, the disease is not a sickle cell disease.

In a preferred embodiment of the methods or embodiments of the invention, the disease is not alzheimer's disease.

In a preferred embodiment of the methods or embodiments of the invention, the disease is not a disease associated with pathological megakaryocyte function.

In a preferred embodiment of the method or embodiment of the invention, the disease is not a disease associated with pathological platelet function.

In the preferred embodiments referenced above, certain disclaimed diseases may be incorporated in any way in the various aspects of the present invention.

In a preferred embodiment of the method of the invention and embodiments thereof, the disease is not a disease selected from the group comprising cancer, cardiovascular disease, systemic lupus erythematosus, sickle cell disease, alzheimer's disease, a disease associated with pathological platelet function and/or a disease associated with pathological megakaryocyte function.

Diseases involved in abnormal platelet function may include post-transfusion purpura (PTP), post-transfusion platelet null (PTPR), Neonatal Alloimmune Thrombocytopenia (NATP), thrombocytopenia and/or essential thrombocytosis.

In a preferred embodiment of the method or embodiment of the invention, the disease-derived nucleic acid is not derived from a megakaryocyte. It was clearly shown that the nucleic acid objects of the invention are taken up or accumulated by thrombocytes from the extracellular (plasma) environment and are not of megakaryocyte lineage origin. Thrombocytes can be analyzed for RNA and/or DNA to detect the presence of RNA and/or DNA of particular disease origin, as demonstrated herein for the detection of the presence of EGFRvIII mutant RNA from glioma.

The present invention describes methods for finding specific nucleic acid transcripts derived from nucleated cells of disease origin within anucleated blood cells, such as thrombocytes extracted from blood. This approach is stable and easy. This is due to the rapid and straightforward extraction procedure and the quality of the extracted NA. In clinical situations, the extraction of thrombocytes (from blood samples) has been achieved in common biological sample collection, and it is therefore foreseen that this process is relatively easy to achieve in clinics.

The present invention provides general methods for analyzing the presence of a disease-derived nucleic acid in the blood of a subject, as well as methods of diagnosing a disease in a subject using the general methods. When the process of the invention is described herein, both embodiments are described.

The method of the invention may be performed on any suitable body sample comprising anucleated blood cells, such as a tissue sample comprising blood, but the sample is preferably whole blood.

A blood sample from a subject may be obtained by any standard method, for example by intravenous drawing.

The amount of blood required is not particularly limited. Depending on the method utilized, the skilled person should be able to establish the required sample size to perform the various steps of the method of the invention and to obtain sufficient NA for genetic analysis. Typically, such amounts should include a volume range from 0.01 μ l to 100 ml.

The body sample may be analyzed immediately after sample collection. Alternatively, the analysis according to the method of the invention may be performed on a stored body sample or a stored part of the anucleated blood cells thereof, preferably thrombocytes. The body sample for testing or a portion of the anucleated blood cells thereof may be preserved using methods and apparatus known in the art. In the collected anucleated blood cell fraction, the thrombocytes preferably remain in an inactivated state (i.e. inactive state). In this way, cellular integrity and disease-derived nucleic acids are best preserved.

In case the fraction of anucleated blood cells is a thrombocyte fraction, the platelet isolated fraction preferably does not comprise platelet poor plasma or Platelet Rich Plasma (PRP). Further separation of platelets is preferred for optimal resolution (resolution).

The body sample may be suitably treated, for example the body sample may be purified, or digested, or specific compounds extracted from the body sample. Depending on the method of characterizing the anucleated blood cells with NA present in said sample, which preferably involves RT-PCR, the anucleated blood cells may be extracted from the sample by methods known to the person skilled in the art and the extracted anucleated blood cells are transferred to any suitable medium for extracting therefrom the NA required for the analytical method. The recipient subject's body sample may be treated to remove excess nucleic acid degrading enzymes (e.g., rnases, dnases) therefrom to prevent early destruction of nucleic acids.

The extraction of thrombocytes from a body sample of a subject may involve any available method. In transfusion medicine, thrombocytes are often collected by plasmapheresis. Plasmapheresis is a medical technique in which the blood of a donor or patient is passed through a device that separates out one particular component and returns the remaining component to the circulatory system. The separation of individual blood components is performed using a dedicated centrifuge. Platelet removal (also known as thrombocyte removal or thrombocyte extraction) is a plasmapheresis process in which thrombocytes are collected. Modern automated platelet removal methods allow blood donors to give them a portion of the thrombocytes while retaining their red blood cells and at least a portion of the plasma. However, as envisioned herein by plasmapheresis, it is possible to provide a body sample that includes thrombocytes, often with easier collection of whole blood and separation of the thrombocyte fraction therefrom by centrifugation. Typically, in such protocols, thrombocytes are first separated from other blood cells by a centrifugation step at about 120 × g for about 20 minutes at room temperature to obtain a platelet rich plasma fraction. The thrombocytes are then washed (e.g., in PBS-EDTA) to remove plasma proteins and to enrich the thrombocytes. The washing step is usually carried out at room temperature of 850 to 1000 Xg for about 10 minutes. Further enrichment may be performed to produce a more pure fraction of thrombocytes.

Platelet isolation typically involves blood sample collection in a Vacutainer tube containing the anticoagulant citric acid (e.g., 36ml citric acid, 5mmol/l KCl, 90mmol/l NaCl, 5mmol/l glucose, 10mmol/l EDTA pH 6.8). Suitable protocols for platelet isolation are described in Ferretti et al (J Clin Endocrinol Metab2002;87: 2180-2184). The method involves a preliminary centrifugation step (1,300 rpm/10 min) to obtain Platelet Rich Plasma (PRP). The platelets were then washed three times in anti-aggregation buffer (Tris-HCl 10mmol/l; NaCl150mmol/l; EDTA1mmol/l; glucose 5mmol/l; pH 7.4) and centrifuged as above to prevent any contamination by plasma proteins and to remove any residual red blood cells. Then, a final centrifugation at 4,000rpm for 20min may be performed to separate platelets. The platelet pellet can be washed, for example, in phosphate buffered saline. To quantitatively determine the level of disease markers, the protein concentration of platelet membranes can be used as an internal reference. Such protein concentrations can be determined by the method of Bradford (Bradford) (Anal Biochem1976;72: 248-254) using serum albumin as a standard.

After a body sample of the subject is prepared and the anucleated blood cells are extracted therefrom, the anucleated blood cells of the subject are screened for the presence of disease-specific nucleic acids. A subject is diagnosed as having a disease as defined herein if a disease-specific nucleic acid is found in the anucleated blood cells of the subject, or if a disease-specific nucleic acid found in the anucleated blood cells of the subject is at a level higher than the anucleated blood cells in an unaffected blood sample of a control subject, which disease-specific nucleic acid is believed to originate from a diseased cell or tissue present in the subject.

Disease-specific nucleic acids (RNA and/or DNA disease markers) are defined as originating from diseased cells that contain or do not contain mutations in nucleic acid sequences associated with or specific for disease; and the disease-specific nucleic acid further comprises a disease-derived anucleated blood cell nucleic acid that is up-regulated or down-regulated relative to nucleic acid in anucleated blood cells from a healthy donor. Thus, the terms "disease-specific nucleic acid" and "disease-derived nucleic acid" are used interchangeably herein. It will be appreciated that non-mutated genes can be identified and used for disease diagnosis. These nucleic acids can be transferred to anucleated blood cells if specific genes are overexpressed in specific diseases. However, if these nucleic acids are already present in the anucleated blood cells of healthy subjects, an increase in the number of nucleic acid copies can be expected in the anucleated blood cells of patients suffering from such diseases. Thus, in particular embodiments of aspects of the invention, quantification of the copy number of a particular gene in anucleated blood cells (e.g., by quantitative PCR or microarray) may be advantageous for detecting the presence of a disease that overexpresses such a gene. Preferably, the disease marker or disease-specific nucleic acid is not derived from a megakaryocyte. In a preferred embodiment of the invention or embodiments thereof, the disease marker or disease-specific nucleic acid is not a mutant at position 12027 of the mitochondrial DNA. In a preferred embodiment of the invention or embodiments thereof, the disease marker or disease-specific nucleic acid is not a mutant at position 11778 of mitochondrial DNA. In a preferred embodiment of the invention or embodiments thereof, the disease marker or disease-specific nucleic acid is not a mutant in the CD109 gene. In a preferred embodiment of the invention or embodiments thereof, the disease marker or disease-specific nucleic acid is not a mutant at position 2108 and/or 954 of the coding region of the CD109 gene. Any of the above disclaimed embodiments may be disclaimed in any combination in aspects herein.

A further step in the method of the invention is to provide a anucleated blood cell-extracted nucleic acid fraction. Such nucleic acid moieties are then used to detect disease markers herein. The anucleated blood cell-extracted nucleic acid fraction can be obtained by any available NA extraction method. RNA extraction is usually performed by using chaotropic agents. The first step in isolating total RNA from cells or tissues is to disrupt the cells under denaturing conditions. In 1979, Chirgwin et al (Biochemistry, 18[24 ]]5294-9, 1979) devised a method for efficient isolation of total RNA by opening the disulfide bonds of proteins by homogenization in a 4M strong protein denaturant, guanidine thiocyanate, with 0.1M 2-mercaptoethanol. The RNA is then isolated by ethanol extraction or by ultracentrifugation over cesium chloride. In 1987, Chomczynski and Sacchi (Analytical Biochemistry,162[1 ]]156, 1987) to design a procedure for rapid single-step extraction using a mixture of guanidinium thiocyanate and phenol-chloroform, particularly for processing large numbers of samples or for isolating RNA from small numbers of cells or tissues. Any commercial kit may also be used for RNA extraction, non-limiting examples of which include Ambion's RNAqueous^TMSystem, Bio101's RNAid Plus kit, Bioline Ltd. ' sRNAce kit, CLONTECH ' sRNA II and NucleoTrap mRNA kit, Invitrogen corp. 's.n.a.p. total RNA isolation kit, and QIAGEN's RNeasy kit.

The disease-derived nucleic acids can be detected in the extracted nucleic acid sample by any available genetic analysis technique that can be adapted to detect mutations in nucleic acid sequences or expression profiles of nucleic acids specific for disease. In general, such sequence mutations can be readily detected by selective nucleic acid hybridization involving the formation of a duplex nucleic acid structure formed by the selective hybridization of two single-stranded nucleic acid sequences to one another. Selective hybridization comprises hybridization of a nucleic acid sequence to a specific nucleic acid sequence of interest under stringent hybridization conditions to a detectable degree (e.g., at least 2-fold above background) above its hybridization to non-nucleic acid sequences of interest, and substantial exclusion of non-nucleic acids of interest. The selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) to each other.

Alternatively, detection of disease-derived nucleic acids can be performed by sequencing techniques (such as DNA and RNA sequencing).

When detecting sequence mutations in an RNA or the expression profile of an RNA, the RNA is preferably transcribed into cDNA prior to detecting sequence mutations therein or quantifying the amount of expression.

RNA can be reverse transcribed into cDNA using RNA-dependent DNA polymerases such as reverse transcriptases from viruses, retrotransposons, bacteria, and the like. These may have RNase H activity, or a reverse transcriptase may be used which is mutated so that the RNase H activity of the reverse transcriptase is limited or not expressed (e.g., MMLV-RT RNase H-). RNA-dependent DNA synthesis (reverse transcription) can also be performed by enzymes that show altered nucleic acid dependence through mutation or modified reaction conditions and thus gain the function of RNA-dependent DNA polymerases.

Once the RNA is reverse transcribed into cDNA, the DNA sequence can be used to analyze for the presence of cancer-specific mutations, or expression profiles can be determined using, for example, selective nucleic acid hybridization as described above. Such techniques are well known in the art and may include selective amplification using amplification primers specific for the mutation to be detected, or selective hybridization to a nucleic acid array using mRNA specific probes. Alternatively, common primers may be used to amplify the DNA including the suspected mutation, which may then be detected in the amplicon by selective nucleic acid hybridization using probes specific for the mutation. Expression profiles are generally obtained using methods of quantitative hybridization well described in the art, examples of which are described in the examples.

In principle, the process of the invention can be carried out by using the following process: any method of nucleic acid amplification, such as the polymerase chain reaction (PCR; Mullis1987, U.S. Pat. Nos. 4,683,195,4,683,202, en4,800,159); or amplification reactions such as ligase chain reaction (LCR; Barany1991, Proc. Natl. Acad. Sci. USA88:189-193; European application No. 320,308), autonomous sequence replication (3 SR; Guatelliet al, 1990, Proc. Natl. Acad. Sci. USA87: 1874-1878), strand displacement amplification (SDA; U.S. Pat. No. 5,270,184, en5,455,166), transcription amplification system (TAS; Kwoh et al, Proc. Natl. Acad. Sci. USA86: 1173-1177), Q-beta replicase (Lizardi et al, 1988, Bio/Technology6: 1197), rolling circle amplification (RCA; U.S. Pat. No. 5,871,921), amplification of gene Nucleic Acid Sequences (NASBA), fragmentation Fragment Length polymorphisms (Cleavase Fragment Length Polymorphism) (U.S. Pat. Polyamplification No. 5,719,028), isothermal amplification primers (for isothermal amplification), and amplification methods for chimeric DNA extension (ICA-25-46597), and other methods suitable for amplification methods.

For amplification of DNA with a small number of mismatches to one or more of the amplification primers, stringency can be reduced (e.g.Using an annealing temperature of 38 ℃ or in the presence of 3.5mM MgCl₂PCR amplification of (1). The skilled person will be able to select conditions of appropriate stringency.

The primers herein may be selected to be "substantially" complementary (i.e., at least 65%, more preferably at least 80% perfect complementarity) to their region of interest present on different strands of each specific sequence to be amplified. Primer sequences comprising, for example, inositol residues or undefined bases, or even primers comprising one or more mismatches when aligned to the sequence of interest, may be used. In general, sequences exhibiting at least 65%, more preferably at least 80% homology to the DNA oligonucleotide sequence of interest are considered suitable for use in the methods of the invention. Sequence mismatches are not critical when using low stringency hybridization conditions.

Detection of the amplification product can in principle be accomplished by any method used in the art. The assay fragment may be directly stained or labeled with a radioactive label, an antibody, a luminescent dye, a fluorescent dye or an enzyme reagent. Direct DNA staining includes, for example, intercalating dyes (such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes).

Alternatively, the DNA fragment may be detected by incorporating labelled dNTP bases into the synthesized DNA fragment. Detection labels that can be associated with the nucleotide bases include, for example, fluorescein, anthocyanin dyes, or BrdUrd.

When a probe-based detection system is used, a suitable detection procedure for use in the present invention may, for example, include an Enzyme Immunoassay (EIA) format (Jacobs et al, 1997, J.Clin.Microbiol.35, 791795). For detection by means of an EIA process, either the forward primer or the reverse primer used in the amplification reaction may comprise a capture group (e.g. a biotin group) for subsequent EIA detection of the DNA amplicon of interest (see below); wherein the biotin group is used for the immobilization of PCR amplicons of the DNA of interest on the wells of e.g.a streptavidin coated microtiter plate. The skilled person will appreciate that other groups of PCR amplicons used to immobilize DNA of interest in an EIA format may be used.

As disclosed herein, the probe for detecting a DNA of interest preferably binds only to at least a portion of a DNA sequence region amplified by a DNA amplification process. One skilled in the art can prepare appropriate probes for detection based on the nucleotide sequence of the DNA of interest without undue experimentation as set forth herein. Furthermore, the complementary sequence of the DNA of interest can be suitably used as a detection probe in the method of the present invention, provided that such a complementary strand is amplified in the amplification reaction used.

Suitable detection procedures for use herein include, for example, the immobilization of amplicons and probing their DNA sequence with a probe, for example by Southern blotting. Other forms may include the EIA form as described above. To facilitate detection of binding, a specific amplicon detection probe may include a label group, such as a fluorophore, chromophore, enzyme, or radiolabel, to facilitate monitoring of binding of the probe to the reaction product of the amplification reaction. Such labels are well known to those skilled in the art and include, for example, Fluorescein Isothiocyanate (FITC), beta-galactosidase, horseradish peroxidase, streptavidin, biotin, digoxigenin,³⁵s or¹²⁵I. Other embodiments will be apparent to those skilled in the art.

The assay can also be performed by the so-called Reverse Line Blot (RLB) assay, as described, for example, by Van den Brule et al (2002, J.Clin. Microbiol.40, 779-787). For this purpose, the 5' amino synthetic RLB probe is preferably used for subsequent immobilization on e.g.carboxyl coated nylon membranes. The advantage of the RLB format is the simplicity of the system and its speed, and thus can be used for high throughput sample processing.

The use of nucleic acid probes for detecting DNA fragments is well known in the art. Most of these procedures involve hybridization of the DNA of interest to a probe, followed by post-hybridization washes. Specificity is usually the effect of post-hybridization washes, the key factors being the ionic strength and temperature of the final wash solutionAnd (4) degree. For DNA-DNA hybrids, T may be used_m(thermal melting point, i.e.at a defined ionic strength and pH, at which approximately 50% of the complementary target sequence hybridizes to a perfectly matched probe) approaches the equation of Meinkoth and Wahl (anal. biochem.,138:267-284 (1984)): t is_m=81.5 ℃ +16.6(log M) +0.41(% GC) -0.61(% formamide) -500/L; where M is the molar concentration of monovalent cations,% GC is the percentage of guanine and cytosine in DNA; % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. For every 1% mismatch T_mAbout 1 ℃ lower, and thus the hybridization and/or wash conditions can be adjusted to hybridize to sequences having the desired identity. For example, if sequences > 90% identical are found, T can be made_mThe reduction is 10 ℃. Generally, stringent conditions are selected to be specific for the sequence and its complement T under defined ionic strength and pH_mAbout 5 deg.c lower. However, very stringent conditions can be used at the ratio T_mHybridizing and/or washing at 1 deg.C, 2 deg.C, 3 deg.C or 4 deg.C; moderately stringent conditions can be used at a ratio T_mHybridization and/or washing at 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C or 10 deg.C; low stringency conditions can be used at ratio T_mHybridization and/or washing is carried out at 11 ℃, 12 ℃,13 ℃,14 ℃,15 ℃ or 20 ℃. Using the equation, hybridization and wash compositions, and the desired T_mOne of ordinary skill in the art will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatch is such that T is_mBelow 45 ℃ (aqueous solution) or 32 ℃ (formamide solution), the SSC concentration is preferably raised so that higher temperatures can be used. For a thorough guidance on Nucleic Acid Hybridization see Tijssen "Biochemical and Molecular Biology Experimental Techniques-Hybridization with Nucleic Acid Probes (Laboratory Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes)," first part, "second part," review of strategies for Hybridization principles and Nucleic Acid Probe assays "(Overview of Hybridization and the strategy of Nucleic Acid Probe assays)," Elsevier Press, New York (1993)), and "Molecular Life" TechniquesChapter ii, edited by Ausubel et al, Greene Publishing and Wiley-Interscience press, new york (1995)).

Preferably, the detection probe is selected to be "substantially" complementary to one strand of a double-stranded DNA amplicon generated by an amplification reaction in the methods of the invention. Preferably, the probe is substantially complementary to the immobilized (e.g., biotin-labeled) antisense strand of the amplicon produced from the DNA of interest.

Allowing the detection probes to contain one or more mismatches to their sequence of interest. In general, sequences exhibiting at least 65%, more preferably at least 80% homology to the DNA oligonucleotide sequence of interest are considered suitable for use in the methods of the invention.

Thus, the step of analyzing the nucleic acid fraction extracted from the anucleated blood cells to determine the presence or absence of a disease marker may be performed by standard nucleic acid analysis techniques. Determining whether there is an alteration in the level of the nucleic acid marker in the nucleic acid fraction relative to the unaffected blood sample should involve a (semi-) quantitative measurement of the amount of the disease marker in the anucleated blood cells. Thus, a highly preferred protocol for detecting disease-specific markers in nucleic acids isolated from anucleated blood cells is quantitative reverse transcription PCR (qRT-PCR) (Freeman et al, BioTechniques26:112-125 (1999)).

As mentioned above, an "unaffected blood sample" refers to the level of a disease marker in a anucleated blood cell of a healthy control subject or from the same subject prior to the onset of disease. Since the characteristics of the anucleated blood cells and the amount of the anucleated blood cell fraction depend on (among other things) the species and age, it is preferred that the non-diseased control anucleated blood cells are from a subject of the same species, age and from the same subpopulation (e.g., smoker/non-smoker). Alternatively, the control data may be obtained from a database and from literature. It is understood that control samples can also be obtained from a subject with a disease at a particular time point to analyze the progression of the disease.

Disease markers include cancer/specific mutations, and cancer-specific mutations can include a variety of known mutations associated with cancer. In thathttp://www.sanger.ac.uk/genetics/CGP/Census/And a non-limiting list of examples of mutations for various cancers is provided in the tables herein.

The present invention further provides a kit for diagnosing a disease in a subject, the kit comprising packaging material comprising at least one reagent for specifically determining the level and/or activity and/or nucleic acid profile of at least one nucleic acid mutant in a sample of anucleated blood cells of a subject. As used herein, the term "diagnosing" refers to determining the presence of a disease, classifying a disease, determining the severity (grade or stage) of a disease, monitoring the progression of a disease, predicting the outcome of a disease and/or the prospects for recovery.

It will be appreciated that the tools necessary for detecting a disease-derived nucleic acid may be provided as a kit, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient for detecting a disease-derived nucleic acid in anucleated blood cells by the method of the present invention.

Alternatively, the kit may comprise separately packaged means for collecting the sample, and primers for specific amplification and/or detection.

The kit may be accompanied by instructions for carrying out the method of the invention.

For example, the kit may be included in a device such as a dipstick (dipstick) or cartridge (cartridge), optionally included in a housing, to which a blood sample or a nucleic acid sample of isolated and/or amplified anucleated blood cells may be applied, and which detects disease-derived or disease-specific nucleic acids or nucleic acid profiles in the sample. The device may comprise any reagent capable of specifically detecting nucleic acids from a disease source. For example, the device can include one or a combination of immobilized mutation-specific hybridization probes that bind to a nucleic acid of disease origin, and an indicator for detecting binding. In embodiments of the invention, a support is provided in the device to which the hybridization probes are removably or fixedly attached.

According to one embodiment, the device may be a lateral flow device comprising access means for flowing a blood sample or an isolated and/or amplified sample of anucleated blood cell nucleic acids into contact with an agent capable of detecting nucleic acids of disease origin. The testing apparatus may also include flow control means for ensuring that the test is functioning correctly. Such flow control means may include control nucleic acids bound to a support that captures detection probes added to the sample as a means of confirming proper flow of sample fluid through the testing device. Alternatively, the flow control means may comprise capture probes in a control zone which captures control nucleic acid naturally present in the sample or to which control nucleic acid is added as a control, again indicating that correct flow is being carried out in the device.

In another aspect, the invention provides the use of a device of the invention to diagnose a disease in a subject using any of the methods described above. Very suitable devices for diagnosing a disease in a subject using any of the above methods include Platelet (Platlet) RNA chips such as those described in Nagalia & Bray (2010) Blood115(1):2-3 and Blood115(1):7-14, such as Gnatenko.

The invention will now be illustrated by way of the following non-limiting examples.

Examples

Example 1

Thrombocytes were isolated from blood samples of 4 glioblastoma patients and 4 healthy donors by a centrifugation step. Then, RNA extraction was performed on thrombocytes using Trizol RNA isolation. The purified thrombocyte RNA samples were then converted to cDNA and analyzed by Agilent (Agilent) 4x44K expression microarray using standard microarray protocols. This enables mRNA expression profiling in different thrombocyte preparations.

In platelets from healthy donors, approximately 8500 RNA transcripts could not be detected by the expression microarray. These transcripts were present at levels below the detection limit of the Agilent4x44K chip in thrombocytes from healthy donors. Thus, such RNAs may all be potential markers for cancer diagnosis. Of the RNAs not detected by expression microarrays in thrombocytes from healthy donors, a significant portion of the RNA was detected in thrombocytes from glioblastoma patients. Table 1 summarizes the unique thrombocyte RNAs detected by expression microarrays in thrombocytes from glioblastoma patients, but not in thrombocytes from healthy donors. The unique RNA transcripts detected in 4/4 patient samples (table 1A) or 3/4 patient samples (table 1B), but not detected in any of the 4 control samples, are summarized in table 1.

Table 1. unique thrombocyte RNA transcripts detected by expression microarrays in thrombocytes from glioblastoma patients, but not in healthy donor thrombocytes.

1A. transcripts detected in thrombocytes in four of the four patient samples, but not in thrombocytes from the control sample.

Transcripts detected in thrombocytes in three of the four patient samples, but not in thrombocytes from the control sample.

TABLE 2 cancer-specific mutations for various cancers.

Code number

Gene ID

Chromosome band

Tumor type (somatic mutation)

Syndrome of cancer

Example 2

Introduction to the design reside in

A diagnostic platform with high predictability in diagnosis, monitoring, and ranking of cancer patients is a key tool in developing personalized medicine. In this example, tumor cells were demonstrated to metastasize (mutated) RNA into platelets (in vitro), and platelets isolated from glioblastoma and prostate cancer patients were shown to contain the cancer-associated RNA biomarkers EGFRvIII and PCA3 and PSA, respectively. Furthermore, gene expression arrays revealed different mRNA signatures in platelets from glioma patients compared to normal control subjects. Because platelets are readily accessible and isolated, they can form an attractive platform for cancer-associated diagnosis.

Method of producing a composite material

Platelet separation and tissue resection

Platelets were separated from whole blood collected in a purple-cap BD Vacutainer containing EDTA anticoagulant by standard centrifugation, and mass (activity and aggregation) and purity were determined by microscopic analysis showing less than 0.1% contamination of red or white blood cells. The separated platelet pellet is then flash frozen for further use. As described in other literature (J.Skog et al, Nat Cell biol.10(12),1470-6 (2008)), at the VU university medical center and in MumeoThe university performed glioma tissue excision and whole blood collection from patients with glioma and prostate cancer.

Microbubble isolation, labeling and transfer

Microvesicles were isolated from U87-EGFRvIII glioma cells and labeled as previously described (j. skog et al, Nat Cell biol.10(12),1470-6 (2008)). After the U87-EGFRvIII microvesicle incubation, the platelets were washed and treated with RNAse enzymes to ensure that EGFRvIII RNA was delivered into the platelets and thus prevent RNAse-mediated degradation. For confocal microscopy analysis, platelets were stained with texas red conjugated wheat germ agglutinin to indicate platelet structure, and microvesicle uptake was analyzed by the presence of green PKH 67. And (4) RNA purification. RNA was isolated using the protocol of miRvana (Ambion) or miRNeasy (Qiagen) according to the manufacturer's instructions. The concentration and quality of RNA were determined using a Bioanalyzer2100 with a total RNA Pico chip (Agilent Corp.).

RT-PCR

RT-PCR of EGFRvIII, PCA3, PSA and GAPDH was performed as described previously (j.skog et al, Nat Cell biol.10(12),1470-6 (2008)) using the following primer sets:

GAPDH primer:

in the forward direction 5'-GAAGGTGAAGGTCGGAGTC-3' of the direction,

and reverse direction 5'-TCAGAAGATGGTGATGGGATTTC-3'.

PSA primer:

in the forward direction 5'-ATGTGGGTCCCGGTTGTCTT-3' of the direction,

and reverse direction 5'-TCCCACAATCCGAGACAGGA-3'.

Nested PCA3 primers:

PCR1:

in the forward direction 5'-AGTCCGCTGTGAGTCT-3' of the direction,

a reverse direction 5'-CCATTTCAGCAGATGTGTGG-3';

PCR2:

in the forward direction 5'-ATCGACGGCACTTTCTGAGT-3' of the direction,

and reverse direction 5'-TGTGTGGCCTCAGATGGTAA-3'.

Nested EGFRvIII primers:

PCR1:

in the forward direction 5'-CCAGTATTGATCGGGAGAGC-3' of the direction,

a reverse direction 5'-TGTGGATCCAGAGGAGGAGT-3';

PCR2:

in the forward direction 5'-GAGCTCTTCGGGGAGCAG-3' of the direction,

reverse direction 5'-GCCCTTCGCACTTCTTACAC-3'

Gene expression array

mRNA expression arrays were performed at the VU university medical center microarray core laboratory (core facility) using the Agilent4x44K gene expression array. The integrity of platelet RNA was assessed using an Agilent2100 bioanalyzer (Agilent technologies, ltd.). RNA samples were labeled using the Agilent Low RNA Input Linear Amplification Kit Plus (Low RNA Input Linear Amplification Kit Plus) (5188-.

Briefly, 25ng of total RNA was amplified and reverse transcribed into cDNA using T7-polymerase, then labeled with Cy3 or Cy 5. Incorporation of the dye was measured using a Nanodrop ND-1000 spectrophotometer. Subsequently, cRNA was hybridized using the Agilent Gene Expression Hybridization Kit (Gene Expression Hybridization Kit) (5188-5242) according to the manufacturer's protocol. Briefly, 825ng of Cy 3-labeled cRNA was mixed with 825ng of Cy 5-labeled cRNA, disrupted at 60 ℃ for 30min in the dark, and hybridized for 17h in a rotary oven at 65 ℃ on Agilent Hybridization Chamber sealing slides (Hybridization Chamber Gasket Slide) (G2534-60011). The slides were scanned using an Agilent Microarray Scanner (Microarray Scanner) (G2565 BA). Image analysis and array normalization were performed using a custom extraction software (feature extraction software) version 9.5 (agilent technologies, ltd.). The Agilent GE2-v5_95 scheme was applied using default settings.

And (5) carrying out statistical analysis.

A heat map (heat map) of gene expression data was generated using a mean centered array (mean centered array) in Excel (Microsoft Office2007 software package) with an s.a.m. analysis plug-in (fig. 3C), with a fixed false discovery rate < 0.5%. The top 30 significantly differentially expressed genes were delineated using the Heatmap Builder v1.1 software (King et al physical genomics. Sep212005;23(1): 103-118)).

Results

In this example, platelets isolated from healthy human control subjects are shown to have the ability to take up RNA-containing microvesicles derived from human brain tumor cells (gliomas) and contain tumor-associated RNA, including mutated EGFRvIII. The uptake of PKH 67-labeled glioma-derived microvesicles in blood platelets was confirmed by FACS analysis and confocal microscopy. In addition, mutant EGFRvIII RNA microvesicle-mediated transfer from healthy control subjects into platelets was shown by RT-PCR. Further, circulating platelets isolated from glioma patients were determined to contain RNA biomarkers (see fig. 3B). RT-PCR was used to determine whether mutated mRNA of EGFRvIII was found in excised high grade glioma tissue (n = 18) and the results were compared to platelets from the same patient and platelets from healthy control subjects (n = 30). Samples were encoded and RT-PCR was performed in a blind test. As previously observed, 4 of the 18 glioma samples (22.5%) contained EGFRvIII transcript. Significantly, EGFRvIII could be amplified from 3 (75%) of the 4 EGFRvIII positive patients and not in platelets of healthy donors (n = 12), but mRNA for GAPDH was detected in all platelet samples. A possible false negative signal was detected in platelets of only one patient, probably due to the blood sample not being adequately processed. In contrast, one patient with an EGFRvIII negative tissue sample was EGFRvIII positive in platelet samples, likely due to heterogeneous distribution of EGFRvIII positive foci in high grade gliomas.

To confirm that the presence of tumor-related information is not unique to platelets from glioma patients, we reported the presence of mRNA encoding the prostate cancer markers PCA3 and PSA in platelets from prostate cancer patients (n = 12), and the absence of mRNA encoding these markers in platelets from healthy control subjects (n = 10) (see fig. 4). Finally, mRNA expression profiles of platelets isolated from healthy control subjects (n = 12) and glioma patients (n = 8) were determined using gene expression arrays. Different mRNA expression profiles were obtained and the identity of the smallest glioma biomarker was detected (fig. 3C, left panel). Interestingly, several potential biomarkers were barely detectable in the control samples, but they were very highly expressed in the glioma samples (fig. 3C, right panel).

In summary, the inventors' findings demonstrate that blood platelets contain cancer markers in the form of tumor-derived or tumor-associated RNA, and thus can be used as a diagnostic platform for molecular expression profiling of cancer in the context of personalized medicine.

Claims (20)

1. A method of analyzing a blood sample of a subject for the presence of a disease marker, the method comprising the steps of:

a) extracting nucleic acid from the anucleated blood cells, preferably thrombocytes, in the blood sample to provide an anucleated blood cells extracted nucleic acid fraction; and

b) analyzing the anucleated blood cells for the presence of a disease marker in the nucleic acid fraction extracted,

wherein the disease marker is a disease-specific mutation in a gene of a nucleated cell of the subject; or

Wherein the disease marker is a disease-specific expression profile of a gene of a nucleated cell of the subject.

2. The method according to claim 1, wherein the anucleated blood cells are thrombocytes or erythrocytes, preferably thrombocytes.

3. The method of claim 1 or 2, wherein the disease is selected from the group consisting of autoimmune diseases, skin diseases, eye diseases, endocrine diseases, neurological disorders, and cardiovascular diseases.

4. The method according to claim 1 or 2, wherein the disease is not a disease selected from the group comprising cancer, cardiovascular disease, systemic lupus erythematosus, sickle cell disease, alzheimer's disease, a disease associated with pathological platelet function, and/or a disease associated with pathological megakaryocyte function.

5. The method according to any of the preceding claims, wherein the anucleated blood cell-extracted nucleic acid fraction is a disease-derived nucleic acid.

6. The method according to any of the preceding claims, wherein the anucleated blood cell-extracted nucleic acid fraction comprises nucleic acids originating from nucleated cells.

7. The method of any one of the preceding claims, wherein the nucleated cells are not megakaryocytes.

8. The method according to any of the preceding claims, wherein the anucleated blood cell-extracted nucleic acid fraction is not megakaryocyte-derived nucleic acid or megakaryocyte-derived RNA.

9. The method of any one of the preceding claims, wherein the disease-specific mutation is located in a chromosomal gene, or wherein the disease-specific expression profile is a disease-specific expression profile of a chromosomal gene.

10. The method according to any of the preceding claims, wherein the nucleic acid is a ribonucleic acid (RNA), preferably an mRNA.

11. The method according to any of the preceding claims, wherein said step b) of analyzing the presence of disease markers in the anucleated blood cell-extracted nucleic acid fraction comprises:

i) selectively amplifying the mutation by polymerase chain reaction amplification of reverse transcriptase using at least one nucleic acid mutation specific amplification primer or probe; or

ii) selectively amplifying the plurality of mRNAs by polymerase chain reaction amplification of reverse transcriptase to determine the expression level of a chromosomal gene encoding the mRNA, thereby providing an expression profile of the gene and comparing the expression profile to a reference profile.

12. The method according to any of the preceding claims, wherein the method is part of a method of diagnosing the disease in a subject, and wherein the presence of the disease marker in the anucleated blood cells-extracted nucleic acid fraction is indicative of the subject having the disease.

13. A method for determining the stage of a disease or the efficacy of a disease treatment in a subject, comprising the steps of:

analyzing a blood sample of a subject for the presence of a disease marker at a first time point using a method according to any one of claims 1 to 12, thereby providing a first value as the level of the disease marker in the subject;

analyzing a blood sample of the subject for the presence of a disease marker at a second time point using the method of any one of claims 1-12, thereby providing a second value as the level of the disease marker in the subject, wherein the subject has been treated for disease between the first time point and the second time point; and

comparing the first value and the second value to determine the efficacy of the disease treatment in the subject.

14. A method for determining the stage of a disease in a subject, comprising the steps of:

analyzing a blood sample of a subject for the presence of a disease marker using a method according to any one of claims 1 to 12, thereby providing a test value as the level of the disease marker in the subject;

providing a reference value for the level of the disease marker, wherein the reference value is associated with a specific stage of disease; and

comparing the test value and the reference value to determine the stage of the disease in the subject.

15. A kit of parts suitable for carrying out the method of any one of claims 1 to 14, the kit comprising packaging material comprising at least one of:

a container for containing anucleated blood cells isolated from a blood sample of a subject;

a reagent for extracting nucleic acids from the anucleated blood cells;

an agent for selectively amplifying, by reverse transcriptase polymerase chain reaction amplification, disease-specific mutations in genes of nucleated cells of the subject from nucleic acids extracted from the anucleated blood cells; and

printed or electronic instructions for carrying out the method of any one of claims 1 to 14;

the kit further comprises:

a reference for the disease marker, wherein the reference indicates the presence or absence of the disease marker in the anucleated blood cell-extracted nucleic acid fraction.

16. The kit of claim 15, wherein the reference is a reference value for the level of nucleic acid comprising the disease-specific mutation in thrombocytes of a healthy control subject or a control subject having the disease; or

Wherein the reference is a reference expression profile for the plurality of mrnas in the anucleated blood cells from a healthy control subject or from a control subject having the disease.

17. The kit of claim 15 or 16, wherein said agent is selected from particles or fluorescent marker labeled anti-anucleated blood cells antibodies, or wherein said instructions are selected from instructions for bead-based anucleated blood cell separation, instructions for FACS sorting of anucleated blood cells, instructions for recovery of anucleated blood cells by centrifugation, or instructions for negative selection of non-anucleated blood cell components.

18. A device for diagnosing a disease, the device comprising a support and at least one reagent attached to the support for specifically determining the level and/or activity of at least one nucleic acid mutant in a anucleated blood cell sample of a subject; and

a computer-readable medium having computer-executable instructions for performing the method of any of claims 1-14.

19. The device of claim 18, wherein the at least one reagent is an oligonucleotide probe or a sequencing primer.

20. The device of claim 18or 19, comprising a lateral flow device, dipstick or cartridge for performing a nucleic acid hybridization reaction between a anucleated blood cell-extracted nucleic acid and at least one nucleic acid mutation-specific amplification primer or oligonucleotide probe specific for a disease-specific mutation; or

For performing a nucleic acid hybridization reaction between the anucleated blood cell-extracted nucleic acids and a plurality of gene-specific amplified primers or a plurality of oligonucleotide probes for providing a disease-specific gene expression profile.