Patent Application
20180037956
The present invention relates to a method, use, kit for diagnosis of a change of the status of health of a subject based on the determination of expression profiles of miRNAs.
Today, biomarkers play a key role in diagnosis, risk stratification, and therapeutic management of various diseases. MicroRNAs (miRNAs) are a new class of biomarkers. They represent a group of small noncoding RNAs that regulate gene expression at the posttranslational level by degrading or blocking translation of messenger RNA (mRNA) targets. It has been found that miRNAs are expressed in a highly tissue-specific manner and further, miRNAs are also present in body fluid samples, including blood. Nevertheless, the mechanism why miRNAs are found in blood, especially in blood cells, or their function in these blood fractions is not understood yet. Especially desirable are non-invasive biomarkers, that allow for quick, easy and cost-effective diagnosis/prognosis, eliminating the need for surgical intervention. Particularly, the potential role of miRNAs as non-invasive biomarkers for general diagnosis of a change in a subjects status of health has not been systematically evaluated yet. Accordingly, there is still a need for effective methods and kits for the non-invasive diagnosis of change in a subjects status of health.
The inventors of the present invention assessed for the first time the expression of miRNAs on a whole-genome level in broad range of subjects being healthy (n=158) of being diagnosed with a plurality of diseases (n=883) including cancer (i.e. lung, colon, kidney, glioblastoma, prostate, melanoma), neurodegenerative (i.e. multiple sclerosis, parkinson), cardiovascular (acute myocardial infarction, heart failure), autoimmune (i.e. sarcoidosis, psoriasis) or inflammatory diseases (i.e. COPD, BPH). They surprisingly found that certain miRNAs (disease-regulated miRNAs,
Thus, said disease-regulated in combination with said disease-preserved miRNAs derived from blood cells or blood cellular fractions comprising erythrocytes, leukocytes and/or thrombocytes are analytically robust and reliable appropriated non-invasive biomarkers with internal normalization capabilities for diagnosis of a change in a subject's status of health using orthogonal diagnostic information originating from the peripheral immune system.
In a first aspect, the invention provides a method for diagnosing the health status in a subject, comprising the steps of:
(a) Determining the expression profile of a first predetermined set of one or more miRNAs in a blood sample from a subject, wherein the miRNAs comprised in said first predetermined set of miRNAs are selected from miRNAs listed in
(b) Determining the expression profile of a second predetermined set of one or more miRNAs in said blood sample from said subject, wherein the miRNAs comprised in said second predetermined set of miRNAs are selected from miRNAs listed in
(c) Normalizing the expression profile of said first predetermined set of miRNAs to said second predetermined set of miRNAs
(d) Comparing the normalized expression profile of step (c) to a reference
(e) Diagnosing a change of the health status in said subject when the comparison to said reference is altered.
In a second aspect, the invention provides a method for diagnosing the health status in a subject, comprising the steps of:
(a) Determining the expression profile of a first predetermined set of miRNAs in a blood sample from a subject
(b) Determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject
(c) Comparing said expression profile of step (a) and said expression profile of step (b) to reference expression profiles
(d) Diagnosing a change of the health status in said subject when the comparison to said first reference expression profile is altered and when the comparison to said second reference expression profile is not altered.
In a third aspect, the invention provides the use of the method according to the first and second aspect of the invention for:
(i.) comparing the health status between one or more subjects,
(ii.) monitoring the health status in a subject,
(iii.) monitoring the state of the immune system in a subject,
(iv.) monitoring the response to therapeutic treatment, preferably drug treatment, in a subject.
In a further aspect of the invention the inventors found that certain miRNAs (cancer-regulated miRNAs,
In a further aspect, the invention provides a method for monitoring the health status in a subject, comprising the steps of:
(a) diagnosing the health status in a subject at a first point in time by carrying out the method according to the first or second aspect,
(b) diagnosing the health status in the subject at one or more later points in time by carrying out the method according to the first or second aspect, and
(c) comparing the health status diagnosed in step (a) with the health status diagnosed in step (b), thereby monitoring the health status in the subject.
This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description.
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used in this specification and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise. For example, the term “a test compound” also includes “test compounds”.
The terms “microRNA” or “miRNA” refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 10 to 33 nucleotides or 15 to 30 nucleotides in length, more preferably of 17 to 27 nucleotides or 18 to 26 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The terms “microRNA*” or “miRNA*” refer to miRNA molecules derived from the passenger strand upon processing. In the context of the present invention, the terms “miRNA” and “miRNA*” are interchangeable used. The miRBase (www.mirbase.org) is a well established repository and searchable database of published miRNA sequences and annotation.
The term “whole blood sample”, as used in the context of the present invention, refers to a blood sample originating from a subject containing all blood fractions, including both the cellular (red blood cells, white blood cells, platelets) and the extra-cellar blood fractions (serum, plasma). The whole blood sample may be derived by removing blood from a subject by conventional blood collecting techniques, but may also be provided by using previously isolated and/or stored blood samples. Preferably, the whole blood sample from a subject (e.g. human or animal) has a volume of between 0.1 and 20 ml, more preferably of between 0.5 and 15 ml, more preferably between 1 and 10 ml and most preferably between 2 and 7.5 ml, i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. Preferably the whole blood sample is collected by means of a blood collection tube, preferably it is collected in a PAXgene Blood RNA tube, in a Tempus Blood RNA tube, in an EDTA-tube (e.g. K2-EDTA Monovette tube), in a Na-citrate tube, Heparin-tube or in a ACD-tube (Acid citrate dextrose). Preferably, when the whole blood sample is collected, the RNA-fraction, especially the miRNA fraction, may be protected/guarded against degradation. For this purpose special collection tubes (e.g. PAXgene Blood RNA tubes from Preanalytix, Tempus Blood RNA tubes from Applied Biosystems) or additives (e.g. RNAlater from Ambion, RNAsin from Promega), that stabilize the RNA fraction and/or the miRNA fraction, may be employed.
The term “blood cell sample”, as used in the context of the present invention, refers to a preparation of the whole blood sample, that comprises or substantially comprises blood cells (red blood cells, white blood cells, platelets), more preferably the blood cell sample contains red blood cells, white blood cells and platelets. Preferably, the blood cell sample does not contain miRNAs that originate from the extra-cellular fraction (e.g. plasma, serum) of whole blood or it does contain miRNAs that originate from the extra-cellular fraction (e.g. plasma, serum) only in minor amounts in order that these do not or do not substantially contribute to the expression profile of the set of one or more miRNA in a blood cell sample according to the present invention. Blood cell samples comprising red blood cells, white blood cells and/or platelets or blood cell samples containing red blood cells, white blood cells and platelets are obtained from processing of whole blood samples collected in PAXgene Blood RNA Tubes, Tempus Blood RNA Tubes, EDTA-tubes (e.g. K2-EDTA Monovette tubes), Na-citrate tubes or Heparin-tubes, maintaining or substantially maintaining the initial cellular distribution (blood cell composition) of the whole blood sample. From the blood cell sample the total RNA (comprising the short RNA fraction including the miRNA fraction) is isolated and which is used for determining the expression profile of a set of at least one, preferably of at least two miRNAs of a subject in said sample.
The term “blood cellular fractions” as used herein relates to cellular fractions, namely blood cell fractions, derived from a blood sample. Blood cell fraction include red blood cells (erythrocytes), white blood cells (leukocytes), platelets (thrombocytes) or subfractions (i.e. Tcells, Bcells, NK-cells, neutrophils, granulocytes, reticulocytes, etc.) of the aforementioned blood cell types thereof or combinations from the aforementioned entities. Preferably, blood cellular fractions do not contain miRNAs that originate from the extra-cellular fraction (e.g. plasma, serum) of whole blood or do contain miRNAs that originate from the extra-cellular fraction (e.g. plasma, serum) only in minor amounts in order that these do not or do not substantially contribute to the expression profile of the set of one or more miRNA in blood cellular fractions according to the present invention. In the context of the present invention, the terms “blood cell sample” and “sample of blood cellular fractions” are interchangeable used.
The term “total RNA” as used herein relates to the isolated RNA comprising the miRNA-fraction present in the respective blood cell sample, which is derived from a whole blood sample. Preferably, the total RNA according to the present invention contains the miRNA-fraction or contains a miRNA-enriched fraction of said total RNA. The total RNA (comprising the miRNA-fraction or miRNA-enriched fraction) is obtained by lysis (e.g. Trizol) of the blood cells in the blood cell sample, followed by RNA purification e.g. by phenol/chloroform extraction and/or separation based techniques (e.g. glass fiber filter column, silica-membrane column). Examples of kits for RNA isolation and purification include the miRNeasy Kits (Qiagen), PAXgene Blood miRNA Kit (Qiagen), mirVana PARIS Kit (Life Technologies), PARIS Kit (Life Technologies), Tempus Spin RNA Isolation Kit (Life Technologies).
The term “disease-regulated miRNAs”, as used herein, refers to at least one fixed defined miRNA comprised in a set which is known to be differential (regulated) between healthy subjects and diseased subjects. Preferred disease-regulated miRNAs are selected from the group of miRNAs listed in
The term “disease-preserved miRNAs”, as used herein, refers to at least one fixed defined miRNA comprised in a set which is known to be not differential (not regulated) between healthy subjects and diseased subjects. Preferred disease-preserved miRNAs are selected from the group of miRNAs listed in
The term “cancer-regulated miRNAs”, as used herein, refers to at least one fixed defined miRNA comprised in a set which is known to be differential (regulated) between healthy subjects and subjects suffering from cancer. Preferred cancer-regulated miRNAs are selected from the group of miRNAs listed in
The term “cancer-preserved miRNAs”, as used herein, refers to at least one fixed defined miRNA comprised in a set which is known to be not differential (not regulated) between healthy subjects and subjects suffering from cancer. Preferred cancer-preserved miRNAs are selected from the group of miRNAs listed in
The term “expression profile” as used in the context of the present invention, refers to the determination of the miRNA expression profile or refers to a measure that correlates with the miRNA expression in a sample (e.g. in a blood cell sample or a blood cellular fraction derived from a whole blood sample). By determining the miRNA expression profile, each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA. The expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche/454 GS FLX), flow cytometry (e.g. LUMINEX, Milipore Guava) and the like, that allow the analysis of miRNA expression profile in a subject and comparison between samples. The sample material measured by the aforementioned means are derived from a blood cell sample and may be a total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the aforementioned RNA/DNA species. The “expression profile”, as used herein, relates to a collection of expression profiles of one or more miRNAs, preferably of least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59 or more miRNAs, preferably selected from miRNAs with SEQ ID NO: 1 to 77, more preferably from miRNAs with SEQ ID NO: 1 to 59.
The term “determining an expression profile in (from) a blood cell sample” as used herein, relates to the determination of the expression profile from the miRNAs present in said blood cell sample, therefore it is a measure that correlates with the miRNAs present in said blood cell sample. Herein, all steps or transformations required to bring the blood cell sample into a form which allows to record the expression profile by any convenient means (e.g. nucleic acid hybridisation, nucleic acid amplification, polymerase extension, mass spectroscopy, flow cytometry, sequencing, next-generation-sequencing) and which are known to the person skilled in the art, are included, e.g. cell-lysis, RNA-isolation, RNA-labeling, polymerase extension of RNA, ligation of RNA reverse-transcription into cDNA, amplification of the cDNA, labelling of cDNA, etc.
The term “diagnosis” as used in the context of the present invention refers to the process of determining or monitoring a possible disease or disorder and therefore is a process attempting to define or monitor the health status of a subject. The determination of the expression profile of one or more miRNAs selected from the list of disease-regulated (
Nucleic acid sequencing may be performed by conventional Sanger sequencing or by so-called “next generation sequencing (NGS)”, which includes—but is not limited to—sequencing-by-synthesis, sequencing-by-ligation, single molecule sequencing, nanopore-sequencing or the like techniques. The NGS may include the following steps: (i) extracting the total RNA from a blood cell sample derived from a whole blood sample of a subject, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using universal or miRNA-specific RT primers (e.g. stem-loop RT primers); (iii) optionally amplifying the obtained cDNA-transcripts (e.g. by PCR), (iv) subjecting the cDNA-transcripts of the miRNAs to the aforementioned sequencing techniques, such as sequencing-by-synthesis, sequencing-by-ligation. In step (ii) single-stranded oligonucleotides are ligated to the 3′-end comprising a universal or miRNA-specific reverse transcriptase (RT)-primer. In case that the cDNA should be amplified in step (iii), the oligo ligated to the 3′-end also comprises a PCT-primer sequence and a single-stranded oligonucleotide is also ligated to the 5′-end, which also comprises a PCR-primer sequence. In step (iii) the cDNA is then amplified making use of the primer sequences introduced in step (ii) at the 3′ and 5′-end.
The term “health status”, as used in the context of the present invention, relates to a measure of the status of the overall health in a subject, including, but not limited to assessment of the presence or absence of an illness or a disease in said subject. According to the present invention, the health status in a subject may be assessed by determining a set of one or more disease-regulated miRNAs in combination with a set of one or more disease-preserved miRNAs from a blood sample, preferably from a blood cell sample or from a blood cellular fraction of said subject. The health status may the presence or absence of a disease in a subject and/or the presence or absence of cancer in a subject. With the methods of the present invention, a change of the health status is diagnosed. Said change of the health status may be an improvement or a worsening of the health status, e.g. the improvement or worsening of a disease and/or the improvement or worsening of cancer.
The term “disease”, as used herein, refers to an abnormal condition that affects the body of a subject. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the subject afflicted, or similar problems for those in contact with the subject. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect subjects not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality. The term “disease”, as used herein, also covers cancer.
The term “infectious disease”, as used herein, refers to any disease which can be transmitted from subject to subject and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), the bird flu, and influenza.
The term “autoimmune disease”, as used herein, refers to any disease in which the body produces an immunogenic (i.e. immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as self and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g. hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g. systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for instance, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
The terms “cancer disease” or “cancer”, as used herein, refer to or describe the physiological condition in a subject that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. The term “cancer” according to the invention also comprises cancer metastases.
The term “subject”, as used in the context of the present invention, means an individual for which the health status is to be assessed, hence a subject may be one that is afflicted by a disease and/or cancer or not, or may be one that is suspected to be afflicted by a disease and/or cancer or not. With the methods of the present invention, a change of the health status is diagnosed. Said change of the health status may be an improvement or a worsening of the health status, e.g. the improvement or worsening of a disease and/or the improvement or worsening of cancer.
The term “therapeutic treatment” relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of a subject. Said treatment may eliminate the disease in a subject, arrest or slow the development of a disease in a subject, inhibit or slow the development of a disease in a subject, decrease the frequency or severity of symptoms in a subject, and/or decrease the recurrence in a subject who currently has or who previously has had a disease.
The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous, unless clearly indicated to the contrary.
In a first aspect, the present invention relates to a method for diagnosing the health status in a subject comprising the steps of:
(a) Determining the expression profile of a first predetermined set of one or more (disease-regulated) miRNAs in a blood sample from a subject, wherein the miRNAs comprised in said first predetermined set of miRNAs are selected from miRNAs listed in
(b) Determining the expression profile of a second predetermined set of one or more (disease-preserved) miRNAs in said blood sample from said subject, wherein the miRNAs comprised in said second predetermined set of miRNAs are selected from miRNAs listed in
(c) Normalizing the expression profile of said first predetermined set of miRNAs to said second predetermined set of miRNAs
(d) Comparing the normalized expression profile of step (c) to a reference
(e) Diagnosing a change of the health status in said subject when the comparison to said reference is altered.
Herein the blood sample is selected from whole blood, blood cells, blood cellular fractions consisting of erythrocytes, leukocytes and thrombocytes, blood cellular fractions comprising erythrocytes, leukocytes and thrombocytes or blood cellular fractions comprising erythrocytes, leukocytes or thrombocytes. Preferably the blood sample is a blood cell sample, more preferably it is a blood cellular fraction comprising erythrocytes, leukocytes and thrombocytes.
According to the present invention the whole blood sample is collected from the subject by conventional blood draw techniques. Blood collection tubes suitable for collection of whole blood include EDTA- (e.g. K2-EDTA Monovette tube), Na-citrate-, ACD-, Heparin-, PAXgene Blood RNA-, Tempus Blood RNA-tubes. According to the present invention the collected whole blood sample, which intermediately may be stored before use, is processed to result in a blood cell sample of whole blood. This is achieved by separation of the blood cell fraction (the cellular fraction of whole blood) from the serum/plasma fraction (the extra-cellular fraction of whole blood), e.g. by centrifugation techniques. It is preferred, that the blood cell sample derived from the whole blood sample comprises red blood cells, white blood cells and/or platelets, it is more preferred that the blood cell sample derived from the whole blood sample comprises red blood cells, white blood cells and platelets.
Preferably, the total RNA, including the miRNA fraction, or the miRNA-fraction is isolated from said blood cells present within said blood cell samples. Kits for isolation of total RNA including the miRNA fraction or kits for isolation of the miRNA-fraction are well known to those skilled in the art, e.g. miRNeasy-kit (Qiagen, Hilden, Germany), Paris-kit (Life Technologies,
Weiterstadt, Germany). The miRNA-profile of first set of one or more disease-regulated miRNAs (selected from the miRNAs listed in
It is preferred that the normalizing in step (c) generates relative expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs. Preferably said relative expression levels are ratios that are obtained from one or more miRNAs comprised is said first set of (disease- and/or cancer-regulated) miRNAs and from one or more miRNAs comprised in said second set of (disease- and/or cancer-preserved) miRNAs from one or more subjects to be diagnosed. More preferably, said relative expression levels are obtained by dividing the expression level of one or more miRNAs comprised is said first set of (disease- and/or cancer-regulated) miRNAs by the level of one or more miRNAs comprised in said second set of (disease- and/or cancer-preserved) miRNAs. Hence, the second set of (disease- and/or cancer-preserved) miRNAs acts as normalizers for said first set of (disease- and/or cancer-regulated) miRNAs. Examples of said relative expression levels are the ratio of the miRNA expression levels of hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-25 (SEQ ID NO:39) or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-192 (SEQ ID NO: 41), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-192 (SEQ ID NO: 41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-874 (SEQ ID NO: 59), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO: 37), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-27b (SEQ ID NO: 34), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-222 (SEQ ID NO:15) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-99b (SEQ ID NO: 54), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-361-3p (SEQ ID NO: 55) from one or more subjects to be diagnosed.
Preferably, the reference to which the normalized expression profile is compared in step (d) is a reference expression profile. It is preferred that the reference expression profile is obtained in a comparable fashion from one or more reference subjects as the expression profiles were obtained from the subjects to be diagnosed. Preferably, the reference is a normalized reference expression profile that is obtained comprising the steps of:
(i.) Determining the expression profile of a first predetermined set of one or more miRNAs in a blood sample from one or more reference subjects, wherein the miRNAs comprised in said first predetermined set of miRNAs are selected from miRNAs listed in
(ii.) Determining the expression profile of a second predetermined set of one or more miRNAs in said blood sample from one or more reference subjects, wherein the miRNAs comprised in said second predetermined set of miRNAs are selected from miRNAs listed in
(iii.) Normalizing the expression profile of said first predetermined set of miRNAs to said second predetermined set of miRNAs said one or more reference subjects.
It is further preferred that the normalizing in step (iii) generates relative reference expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs. Preferably said relative reference expression levels are ratios that are obtained from one or more miRNAs comprised is said first set of (disease- and/or cancer-regulated) miRNAs and from one or more miRNAs comprised in said second set of (disease- and/or cancer-preserved) miRNAs from one or more reference subjects. More preferably, said relative expression levels are obtained by dividing the expression level of one or more miRNAs comprised is said first set of (disease- and/or cancer-regulated) miRNAs by the level of one or more miRNAs comprised in said second set of (disease- and/or cancer-preserved) miRNAs from one or more referenced subjects. Hence, the second set of (disease- and/or cancer-preserved) miRNAs acts as normalizers for said first set of (disease- and/or cancer-regulated) miRNAs. Examples of said relative reference expression levels are the ratio of the miRNA expression levels of hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-140-3p (SEQ ID NO:31), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-425 (SEQ ID NO:32), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-25 (SEQ ID NO:39) or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-192 (SEQ ID NO: 41), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-192 (SEQ ID NO: 41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-874 (SEQ ID NO: 59), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO: 37), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-27b (SEQ ID NO: 34), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-222 (SEQ ID NO:15) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-99b (SEQ ID NO: 54), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-361-3p (SEQ ID NO: 55) from one or more reference subjects.
According to the present invention, the comparison in step (d) is performed using normalized expression levels from a subjects to be diagnosed and normalized reference expression profiles derived from one or more reference subjects which were obtained by employing the same combination of miRNAs, namely the same combination of disease-regulated and disease-preserved miRNAs, more preferably the same ratios of disease-regulated and disease-preserved miRNAs.
Preferably, the comparison in step (d) is by comparing the relative expression levels from said subject to the relative reference expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs.
It is preferred that the relative expression levels are obtained by calculating a ratio from the (disease-regulated) miRNAs listed in
It is also preferred that the relative expression levels are selected from the group of ratios listed in
It is further preferred that in step (e) a change of the health status is diagnosed when the comparison of the relative expression levels from said subject to the relative reference expression levels exceeds a threshold for one or more of said predetermined miRNAs comprised in said set.
Preferably, the threshold for diagnosing a change of the health status is set at 3 standard deviations, preferably is set at 5 standard deviations, more preferably is set at 7 standard deviations.
In a second aspect, the present invention relates to a method for diagnosing the health status in a subject.
It is understood that method according to the second aspect of the invention includes and/or comprises the aspects detailed in the method according to the first aspect of the present invention.
In said second aspect, the present invention relates to a method for diagnosing the health status in a subject comprising the steps of:
(a) Determining the expression profile of a first predetermined set of miRNAs in a blood sample from a subject
(b) Determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject
(c) Comparing said expression profile of step (a) and said expression profile of step (b) to reference expression profiles
(d) Diagnosing a change of the health status in said subject when the comparison to said first reference expression profile is altered and when the comparison to said second reference expression profile is not altered.
Herein, said miRNAs comprised in said first set of predetermined miRNAs and the miRNAs comprised in said first reference expression profile are selected from miRNAs listed in
It is preferred that said blood sample is selected from whole blood, blood cells, blood cellular fractions consisting of erythrocytes, leukocytes and thrombocytes, blood cellular fractions comprising erythrocytes, leukocytes and thrombocytes or blood cellular fractions comprising erythrocytes, leukocytes or thrombocytes. More preferably, the blood sample is a blood cellular fraction comprising erythrocytes, leukocytes and thrombocytes.
It is preferred, that said reference expression profiles according to the first and the second aspect of the invention are derived from an earlier time point from the same subject.
Preferably, the change of the health status according to the first and the second aspect of the invention is an improvement or a worsening of the health status.
Preferably, in the method according to the first or the second aspect of the invention, the nucleic sequences of the at least two miRNAs, namely one disease-regulated miRNAs (of a first predetermined set of one or more miRNAs selected from the miRNAs listed in
It is preferred that the expression profile is determined comprising the steps of:
(a) Providing a whole blood sample of a subject to be diagnosed for the health status
(b) Deriving a blood cell sample from said whole blood sample
(c) Extracting the total RNA from said blood cell sample
(d) Determining the expression profile of first set of one or more disease-regulated miRNAs (selected from the miRNAs listed in
Preferably, the blood cell sample is derived from a whole blood sample by separating the blood cells from the remaining parts of the whole blood sample, which may be achieved by centrifugation, wherein the blood cells (including red blood cells, white blood cells and platelets) form a pellet that may be harvested (see Example 1). The person skilled in the art is aware of alternative methods to separate the blood cells from the remaining parts of the whole blood sample (e.g. size exclusion, size distribution, dielectrophoresis, positive or negative antibody selection etc.).
It is further preferred that the determining of the expression profile includes the reverse-transcription of the nucleotide sequence of the at least one miRNA, preferably at least two miRNAs comprised in the set into cDNA (complementary DNA). Herein, the RNA-sequence is reverse-transcribed into DNA (e.g. by use of reverse-transcriptase) before the expression profile of said miRNAs is determined. Preferably, the nucleotide sequence of the at least one miRNA, preferably at least two miRNAs comprised in the set is reverse-transcribed into cDNA when nucleic acid amplification (PCR, RT-PCR), sequencing (next generation sequencing, Sanger sequencing) or hybridisation based techniques are employed in the determination of the miRNA expression profile. Furthermore, it is preferred that the total RNA is transcribed into cDNA from which the expression profile is determined.
In a further embodiment of the invention according to the first and the second aspect of the invention the disease-regulated miRNAs are cancer-regulated miRNAs, which are selected from the miRNAs listed in
In a third aspect, the invention relates to the use of the method according to the first and the second aspect of the invention.
It is understood that use according to the third aspect of the invention includes and/or comprises the aspects detailed in the method according to the first and second aspect of the present invention.
Said third aspect of the invention relates to the use of the method according to the first and the second aspect of the invention for:
(i.) comparing the health status between one or more subjects,
(ii.) monitoring the health status in a subject,
(iii.) monitoring the state of the immune system in a subject,
(iv.) monitoring the response to therapeutic treatment, preferably drug treatment, in a subject.
In a fourth aspect, the invention relates to a kit for diagnosing the health status of a subject comprising:
It is understood that kit according to the fourth aspect of the invention includes and/or comprises the aspects detailed in the method according to the first and second aspect, the aspects detailed in the use according to the third aspect of the present invention.
Preferably, said means for determining the expression profile comprise:
(i.) a set of at least two polynucleotides for determining of a set comprising at least one miRNA selected from the group consisting of SEQ ID NO: 1 to 26 and at least one miRNA selected from the group consisting of SEQ ID NO: 27 to 59 in a blood sample of a subject, and
(ii.) a microarray, a RT-PCT system, a PCR-system, a flow cytometer, a bead-based multiplex system or a next generation sequencing system.
The kit comprises at least one reference according to the present invention as outlined in the first aspect of the present invention. In a preferred embodiment, the reference may be contained in the data carrier of the kit. In a further preferred embodiment the reference may be a reference sample and/or a reference standard that is included in the kit and which is employed when performing the kit, e.g. in the determining of the expression profile.
Optionally, the kit comprises a data carrier. Preferably the data carrier is an electronic or a non-electronic data carrier, more preferably it is an electronic data carrier, such as a storage medium. The kit optionally comprises a data carrier, which optionally comprises the reference and/or an instruction on how to apply the expression profile and the reference in the diagnosis of the health status. This instruction on how to apply the expression profile and the reference may include instructions for the doctor and/or the diagnostic laboratory that are involved in the diagnosis of the health status. It is preferred that the data carrier further comprises tools for analysis and evaluation of the determined expression profile(s). These tools may be any tools to assist the doctor and/or the diagnostic laboratory in the diagnosis of the health status. It is preferred that the instruction comprised is an algorithm or a software. Preferably, these tools are software-tools that assist in analysis of the determined expression profile(s) and/or assist in the subsequently diagnosis. The tools for analysis and evaluation may include a reference according to the present invention.
The kit optionally comprises a whole blood collection tube, which is preferably selected from group consisting of EDTA-, Na-citrate-, ACD-, Heparin-, PAXgene Blood RNA-, Tempus Blood RNA-tubes and optionally contains an additive for stabilizing the RNA-fraction.
The kit optionally comprises means for deriving the blood cell sample from a whole blood sample. These means are preferably for separating and/or isolating of the respective blood cell sample (e.g. a blood cell sample comprising white blood cells, red blood cells or platelets, a blood cell sample comprising white blood cells, red blood cells and platelets, a platelet-preparation) from the remaining parts of the whole blood sample. These means may include reagents or consumables for isolating/separating the respective blood cell fraction(s) and/or suitable instrumentation (e.g. centrifuge, special collection tubes).
In a further aspect of the invention the inventors found that certain miRNAs (cancer-regulated miRNAs,
In a further aspect, the invention relates to a method for monitoring the health status in a subject, comprising the steps of:
(a) diagnosing the health status in a subject at a first point in time by carrying out the method according to the first or second aspect,
(b) diagnosing the health status in the subject at one or more later points in time by carrying out the method according to the first or second aspect, and
(c) comparing the health status diagnosed in step (a) with the health status diagnosed in step (b), thereby monitoring the health status in the subject.
In one embodiment, the health status in the subject improves or worsens over time.
In one other embodiment, the health status in the subject which is monitored encompasses the state of the immune system of said subject.
In one other embodiment, the subject has received a therapeutic treatment between the first point in time and the one or more later points in time. Said treatment may be a drug treatment, i.e. it may comprise the administration of a drug. Said drug may be a drug to treat a disease and/or cancer in the subject. Said treatment may alternatively and/or additionally comprise surgery, chemotherapy, radiation therapy, and/or immunotherapy.
In summary, the present invention is composed of the following items:
1. A method for diagnosing the health status in a subject, comprising the steps of:
(f) Determining the expression profile of a first predetermined set of one or more miRNAs in a blood sample from a subject, wherein the miRNAs comprised in said first predetermined set of miRNAs are selected from miRNAs listed in
(g) Determining the expression profile of a second predetermined set of one or more miRNAs in said blood sample from said subject, wherein the miRNAs comprised in said second predetermined set of miRNAs are selected from miRNAs listed in
(h) Normalizing the expression profile of said first predetermined set of miRNAs to said second predetermined set of miRNAs
(i) Comparing the normalized expression profile of step (c) to a reference
(j) Diagnosing a change of the health status in said subject when the comparison to said reference is altered
wherein said blood sample is selected from whole blood, blood cells, blood cellular fractions consisting of erythrocytes, leukocytes and thrombocytes, blood cellular fractions comprising erythrocytes, leukocytes and thrombocytes or blood cellular fractions comprising erythrocytes, leukocytes or thrombocytes.
2. The method according to item 1, wherein the normalizing in step (c) generates relative expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs.
3. The method according to item 2, wherein said reference is a normalized reference expression profile that is obtained comprising the steps of:
(i.) Determining the expression profile of a first predetermined set of one or more miRNAs in a blood sample from one or more reference subjects, wherein the miRNAs comprised in said first predetermined set of miRNAs are selected from miRNAs listed in
(ii.) Determining the expression profile of a second predetermined set of one or more miRNAs in said blood sample from one or more reference subjects, wherein the miRNAs comprised in said second predetermined set of miRNAs are selected from miRNAs listed in
(iii.) Normalizing the expression profile of said first predetermined set of miRNAs to said second predetermined set of miRNAs said one or more reference subjects
4. The method according to item 3, wherein the normalizing in step (iii) generates relative reference expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs.
5. The method according to any of the items 1 to 4, wherein the comparison in step (d) is by comparing the relative expression levels from said subject to the relative reference expression levels for each of the miRNAs comprised in said first predetermined set of miRNAs.
6. The method according to any of the items 2, 4 or 5, wherein the relative expression levels are obtained by calculating a ratio from the (disease-regulated) miRNAs listed in
7. The method according to item 6, wherein the relative expression levels are selected from the group of ratios listed in
8. The method according to any of the items 1 to 7, wherein in step (e) a change of the health status is diagnosed when the comparison of the relative expression levels from said subject to the relative reference expression levels exceeds a threshold for one or more of said predetermined miRNAs comprised in said set.
9. The method according to any of the items 1 to 8, wherein said threshold is set at 3 standard deviations, preferably is set at 5 standard deviations, more preferably is set at 7 standard deviations.
10. A method for diagnosing the health status in a subject, comprising the steps of:
(e) Determining the expression profile of a first predetermined set of miRNAs in a blood sample from a subject
(f) Determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject
(g) Comparing said expression profile of step (a) and said expression profile of step (b) to reference expression profiles
(h) Diagnosing a change of the health status in said subject when the comparison to said first reference expression profile is altered and when the comparison to said second reference expression profile is not altered,
(i.) comparing the health status between one or more subjects,
(ii.) monitoring the health status in a subject,
(iii.) monitoring the state of the immune system in a subject, (iv.) monitoring the response to therapeutic treatment, preferably drug treatment, in a subject.
14. The method, use or kit according to any of the items 1 to 13, wherein further miRNAs of said first set of (disease-regulated) miRNAs are selected from the miRNAs listed in
With:
(Within the graphs the annotation of the miRNA-identifiers is truncated due to visualization constraints; herein the relative expression values are as listed above)
The Examples are designed in order to further illustrate the present invention and serve a better understanding. They are not to be construed as limiting the scope of the invention in any way.
Blood of healthy control and diseased subjects PAXgene Blood RNA Tubes (PreAnalytix). For each blood donor 2.5 ml in PAXgene Blood RNA Tubes. The blood cells preparations were derived/obtained from processing the whole blood samples by centrifugation. Herein, the blood cells from the whole blood collected in said blood collection tubes were spun down by 10 min, 5000×g centrifugation. The blood cell pellet (the cellular blood fraction comprising red blood cells, white blood cells and platelets) was harvested for further processing, while the supernatant (including the extra-cellular blood fraction) was discarded. Total RNA, including the small RNA (miRNA-fraction) was extracted from the harvested blood cells using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany); for details see Example 2.
The isolation of total RNA, including the small RNA (miRNA-fraction) was performed by use of the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). Herein, the blood cell pellet (obtained as outlined in Example 1) was thoroughly resuspended in 700 μl QIAzol lysis reagent by pipetting up and down and immediately the suspension was transferred to a new 1.5 ml Eppendorf tube. Then 140 μl chloroform were added, vortexed thoroughly and incubated for 2-3 min at room temperature, followed by centrifugation at 12,000 g for 15 min at 4° C. Afterwards, the upper, aqueous phase was transferred to a new 2 ml tube with great care, without touching the other two phases. Then 1.5 volumes of 100% ethanol were added to the transferred aqueous phase and thoroughly mixing was done by pipetting. 700 μl of sample were then transferred into a column and centrifuged at 13,000 rpm for 15 sec at RT, discarding the flow-through. Afterwards 700 μl of Buffer RWT were added to each column, centrifuged again at 13,000 rpm for 15 sec at RT, discarding the flow-through. Then 500 μl Buffer RPE was added to the column and centrifuged at 13,000 rpm for 15 sec at RT, discarding the flow-through. Afterwards another 500 μl Buffer RPE was added to the column and centrifuged at 13,000 rpm for 2 min at RT, discarding the flow-through. Then the column was placed into a new 2 ml collection tube and centrifuged at 13,000 rpm for 1 min at RT to dry it. The column was transferred into a new 1.5 ml collection tube. For elution of the total RNA incl. microRNA 40 μl RNase-free water was pipetted onto the column and incubated for 1 min, centrifuged at 13.000 rpm at RT for 1 min. Then the eluate was put back onto the same column, incubated for 1 min at RT and centrifuged again for 1 min. The eluted total RNA incl. microRNA was quantified using the NanoDrop 1000 and stored at −20° C. before use in expression profiling experiments. For quality control of the total RNA, 1 μl of total RNA was applied on Agilent's Bioanalyzer, selecting either Agilent's nano- or pico-RNA Chip depending on RNA concentration determined by NanoDrop measurement.
The total RNA-samples including the miRNA-fraction (obtained by protocol Example 2) were analyzed employing microarray hybridization on the Geniom Realtime Analyzer (febit biomed GmbH, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens. Each microfluidic microarray contains complementary dna-probes of 866 miRNAs and miRNA* (each represented by 7 replicates) as annotated in the Sanger miRBase 12.0. Sample labeling with biotine has been carried out by enzymatic on-chip labeling of miRNAs employing febit's MPEA-assay. Following hybridization for 16 hours at 42° C. the biochip was washed automatically and a program for signal enhancement was processed with the Geniom Realtime Analyzer. The resulting detection pictures were evaluated using the Geniom Wizard Software. For each array, the median signal intensity was extracted from the raw data file such that for each miRNA seven intensity values have been calculated corresponding to each replicate copy of miRBase on the array. Following background correction, the seven replicate intensity values of each miRNA were summarized by their median value. To normalize the data across different arrays, quantile normalization was applied and all further analyses were carried out using the normalized and background subtracted intensity values. From median g1 and median g2 the Fold Change of the expression (=qmedian) was calculated as the ratio g1/g2.
After having verified the normal distribution of the measured data, a parametric t-test (unpaired, two-tailed) was carried out for each miRNA separately, to detect miRNAs that show a different behavior in different groups of blood donors. The resulting raw p-values were adjusted for multiple testing by Benjamini-Hochberg adjustment (=ttest_adj). Furthermore, we applied the limma-test for each miRNA separately and corrected according to Benjamini-Hochberg (=limma_adj). Additionally, we applied receiver operating characteristics and calculated the “Area under the Curve”-value (=AUC). The ttest-, limma-test- and AUC-values allow to judge on the statistical significance for each miRNA to be differential expressed between group 1 (g1 subjects) and group 2 (g2 subjects).
The goal of this study was to identify miRNAs, which show a significantly altered expression in different diseases and healthy controls, and miRNAs, which by contrast show very low expression variability in diseases and in controls. The two groups of miRNAs are in the following referred to as “disease-regulated” or “disease-preserved” miRNAs. In total, miRNA profiles have been obtained from 1,049 blood samples all of which were collected in PAXgene Blood RNA tubes using the same protocol. In each case the profiles were generated from the expression values of 848 miRNAs recorded by microarray analysis. To identify “disease-regulated” or “disease-preserved” miRNAs, we used t-test and AUC values. Using 20 different thresholds for p-values or AUC values we obtained different sets of “disease-regulated” (
| Number | Date | Country | Kind |
|---|---|---|---|
| 15157672.5 | Mar 2015 | EP | regional |
| 15158794.6 | Mar 2015 | EP | regional |
| 15164272.5 | Apr 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/053834 | 2/24/2016 | WO | 00 |
