Patent Application
20150045243
The present invention relates to a method for diagnosing and/or prognosing of a disease, preferably lung cancer based on the determination of expression profiles of sets of miRNAs representative for a disease, preferably lung cancer compared to a reference. Furthermore, the present invention relates to sets of polynucleotides and/or primer pairs for detecting sets of miRNAs for diagnosing and/or prognosing of a disease, preferably lung cancer in a biological sample from a subject. Further, the present invention relates to means for diagnosing and/or prognosing of a disease, preferably lung cancer comprising said sets of primer pairs and/or polynucleotides. In addition, the present invention relates to a kit for diagnosing and/or prognosing of a disease, preferably lung cancer comprising means for determining expression profiles of sets of miRNAs representative for a disease, preferably lung cancer and at least one reference. Further, the present invention relates to use of polynucleotides and/or primer pairs for diagnosing and/or prognosing of a disease, preferably lung cancer in a biological samples of a subject. Furthermore, the present invention relates to new miRNA-, miRNA*- and miRNA-precursor sequences identified by next generation sequencing from blood samples.
Today, biomarkers play a key role in early diagnosis, risk stratification, and therapeutic management of various diseases. While progress in biomarker research has accelerated over the last 5 years, the clinical translation of disease biomarkers as endpoints in disease management and as the foundation for diagnostic products still poses a challenge.
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. MiRNAs are important players when it comes to regulate cellular functions and in several diseases, including cancer.
So far, miRNAs have been extensively studied in tissue material. It has been found that miRNAs are expressed in a highly tissue-specific manner. Disease-specific expression of miRNAs have been reported in many human cancers employing primarily tissue material as the miRNA source. In this context miRNAs expression profiles were found to be useful in identifying the tissue of origin for cancers of unknown primary origin.
Since recently it is known that miRNAs are not only present in tissues but also in other body fluid samples, including human blood. Nevertheless, the mechanism why miRNAs are found in body fluids, especially in blood, or their function in these body fluids is not understood yet.
Various miRNA biomarkers found in tissue material have been proposed to be correlated with certain diseases, e.g. cancer. However, there is still a need for novel miRNAs as biomarkers for the detection and/or prediction of these and other types of diseases. Especially desirable are non-invasive biomarkers, that allow for quick, easy and cost-effective diagnosis/prognosis which cause only minimal stress for the patient eliminating the need for surgical intervention
Particularly, the potential role of miRNAs as non-invasive biomarkers for the diagnosis and/or prognosis of a disease, preferably lung cancer has not been systematically evaluated yet. In addition, many of the miRNA biomarkers presently available for diagnosing and/or prognosing of diseases have shortcomings such as reduced sensitivity, not sufficient specificity or do not allow timely diagnosis or represent invasive biomarkers. Accordingly, there is still a need for novel and efficient miRNAs or sets of miRNAs as markers, effective methods and kits for the non-invasive diagnosis and/or prognosis of diseases such as a disease, preferably lung cancer.
The inventors of the present invention assessed for the first time the expression of miRNAs on a whole-genome level in subjects with a disease, preferably lung cancer as non-invasive biomarkers from body fluids, preferably in blood. They surprisingly found novel (previously unknown) miRNAs and miRNAs that are significantly dysregulated in blood of a disease, preferably lung cancer subjects in comparison to healthy controls and thus, miRNAs are appropriated non-invasive biomarkers for diagnosing and/or prognosing of a disease, preferably lung cancer. This finding is surprising, since there is nearly no overlap of the miRNA biomarkers found in blood and the miRNA biomarkers found in tissue material representing the origin of the disease. The inventors of the present invention surprisingly found miRNA biomarkers in body fluids, especially in blood, that have not been found to be correlated to a disease, preferably lung cancer when tissues material was used for this kind of analysis. Therefore, the inventors of the invention identified for the first time miRNAs as non-invasive surrogate biomarkers for diagnosis and/or prognosis of a disease, preferably lung cancer. The inventors of the present invention identified single miRNAs which predict a disease, preferably lung cancer with high specificity, sensitivity and accuracy. The inventors of the present invention also pursued a multiple biomarker strategy, thus implementing sets of miRNA biomarkers for diagnosing and/or prognosing of a disease, preferably lung cancer leading to added specificity, sensitivity, accuracy and predictive power, thereby circumventing the limitations of single biomarker. In detail, by using a machine learning algorithms, they identified unique sets of miRNAs (miRNA signatures) that allow for non-invasive diagnosis of a disease, preferably lung cancer with even higher power, indicating that sets of miRNAs (miRNA signatures) derived from a body fluid sample, such as blood from a subject (e.g. human) can be used as novel non-invasive biomarkers.
Furthermore, the inventors of the present invention identified mayor miRNA (miRNA) or minor miRNAs (miRNA*) or miRNA precursor sequences that were previously unknown and were newly identified by next generation sequencing from blood samples of healthy control subjects and lung cancer patients. These novel miRNAs are suited for diagnosing and/or prognosing of a disease, preferably for diagnosing and/or prognosing cancer, more preferably for diagnosing and/or prognosing lung cancer.
In a first aspect, the invention provides a method for diagnosing and/or prognosing of a disease, preferably lung cancer comprising the steps of:
In a second aspect, the invention provides a set comprising polynucleotides for detecting a set comprising at least two miRNAs for diagnosing and/or prognosing of a disease, preferably lung cancer in a body fluid sample from a subject.
In a third aspect, the invention provides a use of a set of polynucleotides according to the second aspect of the invention for diagnosing and/or prognosing a disease, preferably lung cancer in a subject
In a fourth aspect, the invention provides a set of primer pairs for determining the expression level of a set of miRNAs in a body fluid sample of a subject suffering or suspected of suffering from a disease, preferably lung cancer.
In a fifth aspect, the invention provides a use of set of primer pairs according to the fourth aspect of the invention for diagnosing and/or prognosing a disease, preferably lung cancer in a subject
In a sixth aspect, the invention provides means for diagnosing and/or prognosing of a disease, preferably lung cancer in a body fluid sample of a subject comprising:
In a seventh aspect, the invention provides a kit for diagnosing and/or prognosing of a disease, preferably lung cancer comprising
In an eighth aspect, the invention provides a set of miRNAs in a body fluid sample isolated from a subject for diagnosing and/or prognosing of a disease, preferably lung cancer.
In a ninth aspect, the invention provides a use of a set of miRNAs according to the eighth aspect of the invention for diagnosing and/or prognosing of a disease, preferably lung cancer in a subject,
This summary of the invention does not necessarily describe all features of the invention.
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, not including optionally labels and/or elongated sequences (e.g. biotin stretches). The miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are noncoding RNAs). The genes encoding miRNAs are longer than the processed mature miRNA molecules. The miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70 nucleotide stem-loop structures known as pre-miRNAs in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5′ end. The remaining strand, known as the miRNA*, anti-guide (anti-strand), or passenger strand, is degraded as a RISC substrate. Therefore, the miRNA*s are derived from the same hairpin structure like the “normal” miRNAs. So if the “normal” miRNA is then later called the “mature miRNA” or “guide strand”, the miRNA* is the “anti-guide strand” or “passenger strand”.
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, not including optionally labels and/or elongated sequences (e.g. biotin stretches). The “miRNA*s”, also known as the “anti-guide strands” or “passenger strands”, are mostly complementary to the “mature miRNAs” or “guide strands”, but have usually single-stranded overhangs on each end. There are usually one or more mispairs and there are sometimes extra or missing bases causing single-stranded “bubbles”. The miRNA*s are likely to act in a regulatory fashion as the miRNAs (see also above). In the context of the present invention, the terms “miRNA” and “miRNA*” are interchangeable used. The present invention encompasses (target) miRNAs which are dysregulated in biological samples such as blood or tissue of a disease patients, preferably lung cancer patients in comparison to healthy controls. Said (target) miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to 222.
The term “miRBase” refers to a well established repository of validated miRNAs. The miRBase (www.mirbase.org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download.
As used herein, the term “nucleotides” refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. The term “nucleosides” refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (—CH2-OH), producing nucleotides, which are the molecular building blocks of DNA and RNA.
The term “polynucleotide”, as used herein, means a molecule 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, not including optionally spacer elements and/or elongation elements described below. The depiction of a single strand of a polynucleotide also defines the sequence of the complementary strand. Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The term “polynucleotide” means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. In detail, the polynucleotide may be DNA, both cDNA and genomic DNA, RNA, cRNA or a hybrid, where the polynucleotide sequence may contain combinations of deoxyribonucleotide or ribonucleotide bases, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods.
In the context of the present invention, a polynucleotide as a single polynucleotide strand provides a probe (e.g. miRNA capture probe) that is capable of binding to, hybridizing with, or detecting a target of complementary sequence, such as a nucleotide sequence of a miRNA or miRNA*, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Polynucleotides in their function as probes may bind target sequences, such as nucleotide sequences of miRNAs or miRNAs*, lacking complete complementarity with the polynucleotide sequences depending upon the stringency of the hybridization condition. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence, such as a nucleotide sequence of a miRNA or miRNA*, and the single stranded polynucleotide described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequences are no complementary sequences. The present invention encompasses polynucleotides in form of single polynucleotide strands as probes for binding to, hybridizing with or detecting complementary sequences of (target) miRNAs for diagnosing and/or prognosing of a disease, preferably lung cancer. Said (target) miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to 222.
Because of the conservation of miRNAs among species, for example between humans and other mammals, e.g. animals such as mice, monkey or rat, the polynucleotide(s) of the invention may not only be suitable for detecting a miRNA(s) of a specific species, e.g. a human miRNA, but may also be suitable for detecting the respective miRNA orthologue(s) in another species, e.g. in another mammal, e.g. animal such as mouse or rat.
The term “antisense”, as used herein, refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
The term “label”, as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids at any position, e.g. at the 3′ or 5′ end or internally. The polynucleotide for detecting a miRNA (polynucleotide probe) and/or the miRNA itself may be labeled. For detection purposes, the miRNA(s) or miRNA*(s) may be employed unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind.
The term “stringent hybridization conditions”, as used herein, means conditions under which a first nucleotide sequence (e.g. polynucleotide in its function as a probe for detecting a miRNA or miRNA*) will hybridize to a second nucleotide sequence (e.g. target sequence such as nucleotide sequence of a miRNA or miRNA*), such as in a complex mixture of nucleotide sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength, pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20° C. for short probes (e.g. about 10-35 nucleotides) and up to 60° C. for long probes (e.g. greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.; or 6×SSPE, 10% formamide, 0.01%, Tween 20, 0.1×TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42° C. with wash in 05×SSPE and 6×SSPE at 45° C.
The term “sensitivity”, as used herein, means a statistical measure of how well a binary classification test correctly identifies a condition, for example how frequently it correctly classifies a heart and cardiovascular system disease into the correct type out of two or more possible types (e.g. heart and cardiovascular system disease type and healthy type). The sensitivity for class A is the proportion of cases that are determined to belong to class “A” by the test out of the cases that are in class “A”. A theoretical, optimal prediction can achieve 100% sensitivity (i.e. predict all patients from the sick group as sick).
The term “specificity”, as used herein, means a statistical measure of how well a binary classification test correctly identifies a condition, for example how frequently it correctly classifies a heart and cardiovascular system disease into the correct type out of two or more possible types. The specificity for class A is the proportion of cases that are determined to belong to class “not A” by the test out of the cases that are in class “not A”. A theoretical, optimal prediction can achieve 100% specificity (i.e. not predict anyone from the healthy group as sick).
The term “accuracy”, as used herein, means a statistical measure for the correctness of classification or identification of sample types. The accuracy is the proportion of true results (both true positives and true negatives).
The term “biological sample”, as used in the context of the present invention, refers to any biological sample containing miRNA(s). Said biological sample may be a biological fluid, tissue, cell(s) or mixtures thereof. For example, biological samples encompassed by the present invention are body fluids, tissue (e.g. section or explant) samples, cell culture samples, cell colony samples, single cell samples, collection of single cell samples, blood samples (e.g. whole blood or a blood fraction such as serum or plasma), urine samples, or samples from other peripheral sources. Said biological samples may be mixed or pooled, e.g. a biological sample may be a mixture of blood and urine samples. A “biological sample” may be provided by removing cell(s), cell colonies, an explant, or a section from a subject suspected to be affected by a disease, preferably lung cancer, but may also be provided by using a previously isolated sample. For example, a tissue sample may be removed from a subject suspected to be affected by a disease, preferably lung cancer by conventional biopsy techniques or a blood sample may be taken from a subject suspected to be affected by a disease, preferably lung cancer by conventional blood collection techniques. The biological sample, e.g. tissue or blood sample, may be obtained from a subject suspected to be affected by a disease, preferably lung cancer prior to initiation of the therapeutic treatment, during the therapeutic treatment and/or after the therapeutic treatment.
The term “body fluid sample”, as used in the context of the present invention, refers to liquids originating from the body of a subject. Said body fluid samples include, but are not limited to, blood, urine, sputum, breast milk, cerebrospinal fluid, amniotic fluid, bronchial lavage, colostrum, seminal fluid, cerumen (earwax), endolymph, perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, tears, vaginal secretion, vomit including components or fractions thereof. Said body fluid samples may be mixed or pooled, e.g. a body fluid sample may be a mixture of blood and urine samples or blood and tissue material. A “body fluid sample” may be provided by removing a body liquid from a subject, but may also be provided by using previously isolated sample material.
Preferably, the body fluid 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 10 ml, more preferably between 1 and 8 ml and most preferably between 2 and 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.
In the context of the present invention said “body fluid sample” allows for a non-invasive diagnosis/and or prognosis of a subject.
The term “blood sample”, as used in the context of the present invention, refers to a blood sample originating from a subject. The “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. For example a blood sample may be whole blood, plasma, serum, PBMC (peripheral blood mononuclear cells), blood cellular fractions including red blood cells (erythrocytes), white blood cells (leukocytes), platelets (thrombocytes), or blood collected in blood collection tubes (e.g. EDTA-, heparin-, citrate-, PAXgene-, Tempus-tubes) including components or fractions thereof. For example, a blood sample may be taken from a subject suspected to be affected or to be suspected to be affected by a disease, preferably lung cancer, prior to initiation of a therapeutic treatment, during the therapeutic treatment and/or after the therapeutic treatment.
Preferably, the 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 10 ml, more preferably between 1 and 8 ml and most preferably between 2 and 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.
In the context of the present invention said “body fluid sample” allows for a non-invasive diagnosis/and or prognosis of a subject.
Preferably, when the blood sample is collected from the subject the RNA-fraction, especially the the miRNA fraction, is guarded against degradation. For this purpose special collection tubes (e.g. PAXgene 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 are employed.
The biological sample, preferably the body fluid sample may be from a subject (e.g. human or mammal) that has been therapeutically treated or that has not been therapeutically treated. In one embodiment, the therapeutical treatment is monitored on the basis of the detection of the miRNA or set of miRNAs by the polynucleotide or set of polynucleotides of the invention. It is also preferred that total RNA or a subfraction thereof, isolated (e.g. extracted) from a biological sample of a subject (e.g. human or animal), is used for detecting the miRNA or set of miRNAs by the polynucleotide or set of polynucleotides or primer pairs of the invention.
The term “non-invasive”, as used in the context of the present invention, refers to methods for obtaining a biological sample, particularly a body fluid sample, without the need for an invasive surgical intervention or invasive medical procedure. In the context of the present invention, a blood drawn represents a non-invasive procedure, therefore a blood-based test (utilizing blood or fractions thereof) is a non-invasive test. Other body fluid samples for non-invasive tests are e.g. urine, sputum, tears, mothers mild, cerumen, sweat, saliva, vaginal secretion, vomit, etc.
The term “minimal invasive”, as used in the context of the present invention, refers to methods for obtaining a biological sample, particularly a body fluid sample, with a minimal need for an invasive surgical intervention or invasive medical procedure.
The term “biomarker”, as used in the context of the present invention, represents a characteristic that can be objectively measured and evaluated as an indicator of normal and disease processes or pharmacological responses. A biomarker is a parameter that can be used to measure the onset or the progress of disease or the effects of treatment. The parameter can be chemical, physical or biological.
The term “surrogate biomarker”, as used in the context of the present invention, represents biomarker intended to substitute for a clinical endpoint. It is a measure of a clinical condition or a measure of effect of a certain treatment that may correlate with the real clinical condition (e.g. healthy, diseased) but doesn't necessarily have a guaranteed relationship. An ideal surrogate biomarker is a laboratory substitute for a clinically meaningful result, and should lie directly in the causal pathway linking disease to outcome. Surrogate biomarkers are used when the primary endpoint is undesired (e.g. death). A commonly used example is cholesterol: while elevated cholesterol levels increase the likelihood for heart disease, the relationship is not linear—many people with normal cholesterol develop heart disease, and many with high cholesterol do not.
“Death from heart disease” is the endpoint of interest, but “cholesterol” is the surrogate biomarker.
The term “diagnosis” as used in the context of the present invention refers to the process of determining a possible disease or disorder and therefore is a process attempting to define the (clinical) condition of a subject. The determination of the expression level of a set of miRNAs according to the present invention correlates with the (clinical) condition of a subject. Preferably, the diagnosis comprises (i) determining the occurrence/presence of a disease, preferably lung cancer, (ii) monitoring the course of a disease, preferably lung cancer, (iii) staging of a disease, preferably lung cancer, (iv) measuring the response of a patient with a disease, preferably lung cancer to therapeutic intervention, and/or (v) segmentation of a subject suffering from a disease, preferably lung cancer.
The term “prognosis” as used in the context of the present invention refers to describing the likelihood of the outcome or course of a disease or a disorder. Preferably, the prognosis comprises (i) identifying of a subject who has a risk to develop a disease, preferably lung cancer, (ii) predicting/estimating the occurrence, preferably the severity of occurrence of a disease, preferably lung cancer, and/or (iii) predicting the response of a subject with a disease, preferably lung cancer to therapeutic intervention.
The term “(clinical) condition” (biological state or health state), as used herein, means a status of a subject that can be described by physical, mental or social criteria. It includes so-called “healthy” and “diseased” conditions. For the definition of “healthy” and “diseased” conditions it is referred to the international classification of diseases (ICD) of the WHO (http://www.int/classifications/icd/en/index.html). When one condition is compared according to a preferred embodiment of the method of the present invention, it is understood that said condition is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer. When two or more conditions are compared according to another preferred embodiment of the method of the present invention, it is understood that this is possible for all conditions that can be defined and is not limited to a comparison of a diseased versus healthy comparison and extends to multiway comparison, under the proviso that at least one condition is a disease, preferably lung cancers, preferably a specific form of a disease, preferably lung cancer.
The term “miRNA expression profile” as used in the context of the present invention, represents the determination of the miRNA expression level or a measure that correlates with the miRNA expression level in a biological sample. The miRNA expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray, bead-based methods), 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) and the like, that allow the analysis of differential miRNA expression levels between samples of a subject (e.g. diseased) and a control subject (e.g. healthy, reference sample). The sample material measure by the aforementioned means may be 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. 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 “miRNA expression profile”, as used herein, represents the expression level/expression data of a single miRNA or a collection of expression levels of at least two miRNAs, preferably of least 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 or more, or up to all known miRNAs.
The term “differential expression” of miRNAs as used herein, means qualitative and/or quantitative differences in the temporal and/or local miRNA expression patterns, e.g. within and/or among biological samples, body fluid samples, cells, or within blood. Thus, a differentially expressed miRNA may qualitatively have its expression altered, including an activation or inactivation in, for example, blood from a diseases subject versus blood from a healthy subject. The difference in miRNA expression may also be quantitative, e.g. in that expression is modulated, i.e. either up-regulated, resulting in an increased amount of miRNA, or down-regulated, resulting in a decreased amount of miRNA. The degree to which miRNA expression differs need only be large enough to be quantified via standard expression characterization techniques, e.g. by quantitative hybridization (e.g. to a microarray, to beads), 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 FL), flow cytometry (e.g. LUMINEX) and the like.
Nucleic acid hybridization may be performed using a microarray/biochip or in situ hybridization. In situ hybridization is preferred for the analysis of a single miRNA or a set comprising a low number of miRNAs (e.g. a set of at least 2 to 50 miRNAs such as a set of 2, 5, 10, 20, 30, or 40 miRNAs). The microarray/biochip, however, allows the analysis of a single miRNA as well as a complex set of miRNAs (e.g. a all known miRNAs or subsets thereof).
For nucleic acid hybridization, for example, the polynucleotides (probes) according to the present invention with complementarity to the corresponding miRNAs to be detected are attached to a solid phase to generate a microarray/biochip (e.g. 222 polynucleotides (probes) which are complementary to the 222 miRNAs having SEQ ID NO: 1 to 222. Said microarray/biochip is then incubated with a biological sample containing miRNAs, isolated (e.g. extracted) from the body fluid sample such as blood sample from a subject such as a human or an animal, which may be labelled, e.g. fluorescently labelled, or unlabelled. Quantification of the expression level of the miRNAs may then be carried out e.g. by direct read out of a label or by additional manipulations, e.g. by use of a polymerase reaction (e.g. template directed primer extension, MPEA-Assay, RAKE-assay) or a ligation reaction to incorporate or add labels to the captured miRNAs.
Alternatively, the polynucleotides which are at least partially complementary (e.g. a set of chimeric polynucleotides with each a first stretch being complementary to a set of miRNA sequences and a second stretch complementary to capture probes bound to a solid surface (e.g. beads, Luminex beads)) to miRNAs having SEQ ID NO: 1 to 222. are contacted with the biological sample containing miRNAs (e.g a body fluid sample, preferably a blood sample) in solution to hybridize. Afterwards, the hybridized duplexes are pulled down to the surface (e.g a plurality of beads) and successfully captured miRNAs are quantitatively determined (e.g. FlexmiR-assay, FlexmiR v2 detection assays from Luminex).
Nucleic acid amplification may be performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative polymerase chain reaction (RT qPCR). The standard real time polymerase chain reaction (RT-PCR) is preferred for the analysis of a single miRNA or a set comprising a low number of miRNAs (e.g. a set of at least 2 to 50 miRNAs such as a set of 2, 5, 10, 20, 30, or 40 miRNAs), whereas high-throughput RT-PCR technologies (e.g. OpenArray from Applied Biosystems, SmartPCR from Wafergen, Biomark System from Fluidigm) are also able to measure large sets (e.g a set of 10, 20, 30, 50, 80, 100, 200 or more) to all known miRNAs in a high parallel fashion. RT-PCR is particularly suitable for detecting low abandoned miRNAs.
The aforesaid real time polymerase chain reaction (RT-PCR) may include the following steps:
(i) extracting the total RNA from a biological sample or body fluid sample such as a blood sample (e.g. whole blood, serum, or plasma) of a subjects such as human or animal, and obtaining cDNA samples by RNA reverse transcription (RT) reaction using universal or miRNA-specific primers; or collecting a body fluid sample such as urine or blood sample (e.g. whole blood, serum, or plasma) of a patient such as human or animal, and conducting reverse transcriptase reaction using universal or miRNA-specific primers (e.g. looped RT-primers) within the body fluid sample such as urine or blood sample (e.g. whole blood, serum, or plasma) being a buffer so as to prepare directly cDNA samples, (ii) designing miRNA-specific cDNA forward primers and providing universal reverse primers to amplify the cDNA via polymerase chain reaction (PCR), (iii) adding a fluorescent dye (e.g. SYBR Green) or a fluorescent probe (e.g. Taqman probe) probe to conduct PCR, and (iv) detecting the miRNA(s) level in the body fluid sample such as urine or blood sample (e.g. whole blood, serum, or plasma).
A variety of kits and protocols to determine an expression profile by real time polymerase chain reaction (RT-PCR) such as real time quantitative polymerase chain reaction (RT qPCR) are available. For example, reverse transcription of miRNAs may be performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer's recommendations. Briefly, miRNA may be combined with dNTPs, MultiScribe reverse transcriptase and the primer specific for the target miRNA. The resulting cDNA may be diluted and may be used for PCR reaction. The PCR may be performed according to the manufacturer's recommendation (Applied Biosystems). Briefly, cDNA may be combined with the TaqMan assay specific for the target miRNA and PCR reaction may be performed using ABI7300. Alternative kits are available from Ambion, Roche, Qiagen, Invitrogen, SABiosciences, Exiqon etc.
The term “subject”, as used in the context of the present invention, means a patient or individual or mammal suspected to be affected by a disease, preferably lung cancer. The patient may be diagnosed to be affected by a disease, preferably lung cancer, i.e. diseased, or may be diagnosed to be not affected by a disease, preferably lung cancer, i.e. healthy. The subject may also be diagnosed to be affected by a specific form of a disease, preferably lung cancer. The subject may further be diagnosed to develop a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer as the inventors of the present invention surprisingly found that miRNAs representative for a disease, preferably lung cancer are already present in the biological sample, e.g. blood sample, before a disease, preferably lung cancer occurs or during the early stage of a disease, preferably lung cancer. It should be noted that a subject that is diagnosed as being healthy, i.e. not suffering from a disease, preferably lung cancer or from a specific form of a disease, preferably lung cancer, may possibly suffer from another disease not tested/known. The subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human subjects are particularly preferred. Therefore, the miRNA from a subject may be a human miRNA or a miRNA from another mammal, e.g. an animal miRNA such as a mouse, monkey or rat miRNA, or the miRNAs comprised in a set may be human miRNAs or miRNAs from another mammal, e.g. animal miRNAs such as mouse, monkey or rat miRNAs.
The term “control subject”, as used in the context of the present invention, may refer to a subject known to be affected with a disease, preferably lung cancer (positive control), i.e. diseased, or to a subject known to be not affected with a disease, preferably lung cancer (negative control), i.e. healthy. It may also refer to a subject known to be effected by another disease/condition (see definition “(clinical) condition”). It should be noted that a control subject that is known to be healthy, i.e. not suffering from a disease, preferably lung cancer, may possibly suffer from another disease not tested/known. The control subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human “control subjects” are particularly preferred.
The term “set comprising at least two miRNAs representative for a disease, preferably lung cancer”, as used herein, refers to refers to at least two fixed defined miRNAs comprised in a set which are known to be differential between subjects (e.g. humans or other mammals such as animals) suffering from a disease, preferably lung cancer (diseased state) and control subjects (e.g. humans or other mammals such as animals and are, thus, representative for a disease, preferably lung cancer. Said “set comprising at least two miRNAs representative for a disease, preferably lung cancer” are preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
The term “lung cancer”, referrers to carcinoma that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer. Lung cancer may be seen on chest radiograph and computed tomography (CT scan). The diagnosis is confirmed with a biopsy. This is usually performed by bronchoscopy or CT-guided biopsy. Treatment and prognosis depend on the histological type of cancer, the stage (degree of spread), and the patient's general wellbeing, measured by performance status. Common treatments include surgery, chemotherapy, and radiotherapy. NSCLC is sometimes treated with surgery, whereas SCLC usually responds better to chemotherapy and radiation therapy. The non-small-cell lung carcinomas (NSCLC) are grouped together because their prognosis and management are similar. There are three main sub-types: squamous cell lung carcinoma, adenocarcinoma, and large-cell lung carcinoma.
Accounting for 25% of lung cancers, squamous cell lung carcinoma usually starts near a central bronchus. A hollow cavity and associated necrosis are commonly found at the center of the tumor. Well-differentiated squamous cell lung cancers often grow more slowly than other cancer types. Adenocarcinoma accounts for 40% of non-small-cell lung cancers. It usually originates in peripheral lung tissue. Most cases of adenocarcinoma are associated with smoking; however, among people who have never smoked (“never-smokers”), adenocarcinoma is the most common form of lung cancer. A subtype of adenocarcinoma, the bronchioloalveolar carcinoma, is more common in female never-smokers, and may have different responses to treatment.
Due to the shortcomings of current state of the art diagnosis for a disease, preferably lung cancer, there is an urgent need for better, non-invasive tests to further diagnosis and prognosis options for patients.
Due to the shortcomings of current state of the art diagnosis for a disease, preferably lung cancer, there is an urgent need for better, non. invasive tests to further diagnosis and prognosis options for patients.
The inventors of the present invention surprisingly found that miRNAs are significantly dysregulated in body fluid samples such as blood of a disease, preferably lung cancer subjects in comparison to a cohort of controls (healthy subjects) and thus, miRNAs are appropriated biomarkers for diagnosing and/or prognosing of a disease, preferably lung cancer in a non-invasive fashion or minimal-invasive fashion. Furthermore, the sets of miRNAs of the present invention lead to high performance in diagnosing and/or prognosing of a disease, preferably lung cancer, thus expose very high specificity, sensitivity and accuracy. They succeeded in determining the miRNAs that are differentially regulated in body fluid samples from patients having a disease, preferably lung cancer compared to a cohort of controls (healthy subjects) (see experimental section for experimental details). Additionally, the inventors of the present invention performed hypothesis tests (e.g. t-test, limma-test) or other measurements (e.g. AUC, mutual information) on the expression level of the found miRNAs, in all controls (healthy subjects) and subjects suffering from a disease, preferably lung cancer. These tests resulted in a significance value (p-value) for each miRNA. This p-value is a measure for the diagnostic power of each of these single miRNAs to discriminate, for example, between the two clinical conditions: controls (healthy subjects), i.e. not suffering from a disease, preferably lung cancer, or diseased, i.e. suffering from a disease, preferably lung cancer. Since a manifold of tests are carried out, one for each miRNA, the p-values may be too optimistic and, thus, over-estimate the actual discriminatory power. Hence, the p-values are corrected for multiple testing by the Benjamini Hochberg approach.
An overview of the miRNAs that are found to be significantly differentially regulated in blood samples of lung cancer patients is provided in
Usually the diagnostic power of a single miRNA biomarker is not sufficient to reach high accuracy, specificity and sensitivity for discrimination between healthy subjects (controls) and subjects suffering from a disease, preferably lung cancer, hence no simple threshold method can be used for diagnosis and/or prognosis.
Therefore, the inventors of the present invention employed more than one miRNA biomarker, i.e. sets of miRNA biomarkers (signatures), to further increase and/or improve the performance for diagnosing and/or prognosing of subjects suffering from a disease, preferably lung cancer. This leads to a significant increase in sensitivity, specificity and accuracy when compared to the prior art.
In order to be able to discriminate, for example, between two or more clinical conditions, e.g. healthy and suffering from a disease, preferably lung cancer, for a defined set of miRNA biomarkers, the inventors of the present invention applied a machine learning approach (e.g. t-test, AUC, WMW, support vector machine, hierarchical clustering, or k-means) which leads to an algorithm that is trained by reference data (i.e. data of reference miRNA expression profiles from the two clinical conditions, e.g. healthy and suffering from a disease, preferably lung cancer, for the defined set of miRNA markers) to discriminate between the two statistical classes (i.e. two clinical conditions, e.g. healthy or suffering from a disease, preferably lung cancer).
The inventors of the present invention surprisingly found that this approach yields in miRNAs or miRNA sets (signatures) that provide high diagnostic accuracy, specificity and sensitivity in the determination of a disease, preferably lung cancer in patients (see
An exemplarily approach to arrive at miRNA sets/signatures that correlate with a disease, preferably lung cancer is summarized below:
In a first aspect, the present invention relates to a method for diagnosing and/or prognosing of a disease, comprising the steps of:
It is preferred that the body fluid sample is a blood sample, particularly preferred it is a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred it is whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
Preferably, the set comprising at least two miRNAs is from the group consisting of SEQ ID NO: 1 to 222
Preferably the disease to be diagnosed or prognosed is lung cancer, particularly preferred the disease is non-small-cell lung carcinoma (NSCLC).
When diagnosing and/or prognosing lung cancer it is preferred that further nucleotide sequences of the miRNAs comprised in the set are selected from the group consisting of SEQ ID NO: 223 to 254, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is preferred that in the method according to the first aspect of the invention at least one nucleotide sequences of the miRNAs comprised in the set is selected from the group consisting of SEQ ID NO: 1 to 12, a fragment thereof, and a sequence having at least 90% sequence identity thereto. It is particularly preferred that at least one nucleotide sequences of the miRNAs comprised in the set is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, a fragment thereof, and a sequence having at least 90% sequence identity thereto.
It is particularly preferred in the method according to the first aspect of the invention that the expression of the miRNA with nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 4, 5, 6 is upregulated in the disease in comparison to the reference (
It is further particularly preferred in the method according to the first aspect of the invention that the expression of the miRNA with nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 7, 8, 9, 10, 11, 12 is downregulated in the disease in comparison to the reference (
Thus, it is preferred that the method for diagnosing and/or prognosing of a disease comprises the steps of:
Thus, for analysis of a body fluid sample (e.g. blood sample) in step (i) of the method of the present invention, an expression profile of a set comprising at least two miRNAs which are known to be differential between subjects (e.g. humans or other mammals such as animals) having or being suspected to have a disease, preferably lung cancer or a special form of a disease, preferably lung cancer (diseased state) and subjects (e.g. humans or other mammals such as animals) not having a disease, preferably lung cancer or a special form of a disease, preferably lung cancer (healthy/control state) and are, thus, representative for a disease, preferably lung cancer, is determined, wherein the nucleotide sequences of said miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is more particularly preferred that an expression profile of a set comprising, essentially consisting of, or consisting of at least 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 or more, or comprising/consisting of 222 miRNAs, representative for a disease, preferably lung cancer in a body fluid sample (e.g. a blood sample) from a subject (e.g. a human or another mammal such as an animal) is determined in the step (i) of the method of the present invention, wherein the nucleotide sequences of said miRNAs are selected from the group consisting of
Additionally, it is more particularly preferred that an expression profile of a set comprising, essentially consisting of, or consisting of at least 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 or more, or comprising/consisting of 222 miRNAs, representative for a disease, preferably lung cancer in a body fluid sample (e.g. a blood sample) from a subject (e.g. a human or another mammal such as an animal) is determined in the step (i) of the method of the present invention, wherein the set comprising at least two miRNAs is selected from the group consisting of
It is particularly preferred that the nucleotide sequences as defined in (iv) have at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides, preferably over the whole length, to the nucleotide sequences of the nucleotides according to (i) or (ii) or nucleotide fragments according to (iii).
Furthermore, according to the present invention, a first diagnosis and/or prognosis of a disease, preferably lung cancer can be performed employing, as disclosed, miRNA-detection in a body fluid sample, e.g. in blood, followed by a second diagnosis and/or prognosis that is based on other methods (e.g. other biomarkers and/or imaging methods).
Furthermore, according to the present invention, the set comprising at least two miRNAs for diagnosing and/or prognosing a disease, preferably lung cancer in a body fluid sample, e.g. blood sample, from a patient, e.g. human or animal, may be established on one experimental platform (e.g. microarray/biochip), while for routine diagnosis/prognosis another experimental platform (e.g. qPCR) may be chosen.
Subsequent to the determination of an expression profile (of expression profile data) of a set comprising at least two miRNAs representative for a disease, preferably lung cancer as defined above in a body fluid sample (e.g. blood sample) from a patient (e.g. human or animal) in step (i) of the method for diagnosing and/or prognosing of a disease, preferably lung cancer of the present invention, said method further comprises the step (ii) of comparing said expression profile (expression profile data) to a reference, wherein the comparison of said expression profile (expression profile data) to said reference allows for the diagnosis and/or prognosis of a disease, preferably lung cancer.
The subject (e.g. human or another mammal (e.g. animal)) to be diagnosed with the method of the present invention may suffer, may be suspected to suffer or may not suffer from a disease, preferably lung cancer. The subject (e.g. human or another mammal (e.g. animal)) to be diagnosed with the method of the present invention may suffer from a specific type of a disease, preferably lung cancer. It is also possible to determine, whether the subject (e.g. human or another mammal (e.g. animal) to be diagnosed will develop a disease, preferably lung cancer as the inventors of the present invention surprisingly found that miRNAs representative for a disease, preferably lung cancer are already present in the body fluid sample, e.g. blood sample, before a disease, preferably lung cancer occurs or during the early stage of a disease, preferably lung cancer.
The reference may be the reference (e.g. reference expression profile (data)) of a healthy condition (i.e. not a disease, preferably lung cancer), may be the reference (e.g. reference expression profile (data)) of a diseased condition (i.e. a disease, preferably lung cancer) or may be the reference (e.g. reference expression profiles (data)) of at least two conditions from which at least one condition is a diseased condition (i.e. a disease, preferably lung cancer). For example, (i) one condition may be a healthy condition (i.e. not a disease, preferably lung cancer) and one condition may be a diseased condition (i.e. a disease, preferably lung cancer), or (ii) one condition may be a diseased condition (e.g. a specific form of a disease, preferably lung cancer) and one condition may be another diseased condition (i.e. specific form of a disease, preferably lung cancer, or other timepoint of treatment, other therapeutic treatment).
Further, the reference may be the reference expression profiles (data) of essentially the same, preferably the same, set of miRNAs of step (i), preferably in a body fluid sample originated from the same source (e.g. urine, or blood such as serum, plasma, or blood cells) as the body fluid sample from the subject (e.g. human or animal) to be tested, but obtained from subjects (e.g. human or animal) known to not suffer from a disease, preferably lung cancer and from subjects (e.g. human or animal) known to suffer from a disease, preferably lung cancer (e.g. a disease, preferably lung cancer, e.g. a disease, preferably lung cancer that has been therapeutically treated).
Preferably, both the reference expression profile and the expression profile of step (i) are determined in the same body fluid sample, e.g. urine, or blood sample including a whole blood, a blood serum sample, blood plasma sample or a blood cell sample (e.g. erythrocytes, leukocytes and/or thrombocytes). It is understood that the reference expression profile is not necessarily obtained from a single subject known to be affected by a disease, preferably lung cancer or known to be not affected by a disease, preferably lung cancer (e.g. healthy subject, such as healthy human or animal, or diseased subject, such as diseased human or animal) but may be an average reference expression profile of a plurality of subjects known to be affected by a disease, preferably lung cancer or known to be not affected by a disease, preferably lung cancer (e.g. healthy subjects, such as healthy humans or animals, or diseased subjects, such as diseased humans or animals), e.g. at least 2 to 200 subjects, more preferably at least 10 to 150 subjects, and most preferably at least 20 to 100 subjects, (e.g. healthy subject, such as healthy human or animal, or diseased subject, such as diseased human or animal). The expression profile and the reference expression profile may be obtained from a subject/patient of the same species (e.g. human or animal), or may be obtained from a subject/patient of a different species (e.g. human or animal). Preferably, the expression profile is obtained from a subject known to be affected by a disease, preferably lung cancer or known to be not affected by a disease, preferably lung cancer of the same species (e.g. human or animal), of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. infant, young child, juvenile, adult) as the subject (e.g. human or animal) to be tested or diagnosed.
Thus, in a preferred embodiment of the method of the present invention, the reference is a reference expression profile (data) of at least one subject, preferably the reference is an average expression profile (data) of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects, with one known clinical condition which is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer, or which is not a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer (i.e. healthy/healthiness), wherein the reference expression profile is the the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs of step (i). Preferably, the reference expression profile is the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto of step (i).
The comparison of the expression profile of the patient to be diagnosed (e.g. human or animal) to the (average) reference expression profile may then allow for diagnosing and/or prognosing of a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer (step (ii)), either the subject/patient (e.g. human or animal) to be diagnosed is healthy, i.e. not suffering from a disease, preferably lung cancer, or diseased, i.e. suffering from a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer.
The comparison of the expression profile of the subject (e.g. human or animal) to be diagnosed to said reference expression profile(s) may then allow for the diagnosis and/or prognosis of a disease, preferably lung cancer (step (ii)), either the subject (e.g. human or animal) to be diagnosed is healthy, i.e. not suffering from a disease, preferably lung cancer, or the subject (e.g. human or animal) is diseased, i.e. suffering from a disease, preferably lung cancer.
The comparison of the expression profile of the patient (e.g. human or animal) to be diagnosed to said reference expression profiles may then allow for the diagnosis/prognosis of a specific form of a disease, preferably lung cancer (step (ii)), e.g. whether the patient to be diagnosed suffers from a disease, preferably lung cancer.
In a particularly preferred embodiment of the method of the present invention, the reference is an algorithm or mathematical function. Preferably, the algorithm or mathematical function is obtained on the basis of reference expression profiles (data) of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects, with two known clinical conditions from which one is a disease, preferably lung cancer, wherein the reference expression profiles is the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs of step (i). Preferably, is the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto of step (i).
It is preferred that the algorithm or mathematical function is obtained using a machine learning approach.
Machine learning approaches may include but are not limited to supervised or unsupervised analysis: classification techniques (e.g. naïve Bayes, Linear Discriminant Analysis, Quadratic Discriminant Analysis Neural Nets, Tree based approaches, Support Vector Machines, Nearest Neighbour Approaches), Regression techniques (e.g. linear Regression, Multiple Regression, logistic regression, probit regression, ordinal logistic regression ordinal Probit-Regression, Poisson Regression, negative binomial Regression, multinomial logistic Regression, truncated regression), Clustering techniques (e.g. k-means clustering, hierarchical clustering, PCA), Adaptations, extensions, and combinations of the previously mentioned approaches.
The inventors of the present invention surprisingly found that the application/use of a machine learning approach (e.g. t-test, AUC, support vector machine, hierarchical clustering, or k-means) leads to the obtainment of an algorithm or mathematical function that is trained by the reference expression profile(s) or reference expression profile data mentioned above (e.g. trained by the miRNA reference expression profile (data) of a diseased condition (i.e. a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer), for example, obtained from subjects (e.g. humans or animals) known to suffer from a disease, preferably lung cancer or from a specific form of a disease, preferably lung cancer (i.e. being diseased) and/or a trained by the miRNA reference expression profile (data) of a healthy condition (i.e. not a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer), for example, obtained from subjects (e.g. humans or animals) known to not suffer from a disease, preferably lung cancer or from a specific form of a disease, preferably lung cancer and that this allows a better (i) discrimination between the at least two (e.g. 2 or 3) clinical conditions (the at least two statistical classes, e.g. the two conditions healthy or suffering from a disease, preferably lung cancer or the two clinical conditions suffering from a specific form of a disease, preferably lung cancer or suffering from another specific form of a disease, preferably lung cancer or at least three clinical conditions, e.g. the three clinical conditions healthy, suffering from a specific form of a disease, preferably lung cancer or suffering from another specific form of a disease, preferably lung cancer or (ii) decision whether the at least one clinical condition (the one condition healthy or suffering from a disease, preferably lung cancer is present. In this way, the performance for diagnosing/prognosing of individuals suffering from a disease, preferably lung cancer can be increased (see also experimental section for details).
Thus, in a preferred embodiment of the method of the present invention, the algorithm or mathematical function is obtained using a machine learning approach, wherein said algorithm or mathematical function is trained by a reference expression profile (data) of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects with two known clinical condition for which one is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer, wherein the reference expression profile is the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs of step (i), preferably to decide whether the at least one clinical condition which is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer.
Further, for instance, the machine learning approach may be applied to the reference expression profiles (data) of a set comprising at least 2 miRNAs (e.g. 10 miRNAs such as miRNAs according to SEQ ID NO: 1 to 10) of at least one subject (e.g. human or animal) known to suffer from a disease, preferably lung cancer and of at least one subject (e.g. human or animal) known to be healthy and may led to the obtainment of an algorithm or mathematical function. This algorithm or mathematical function may then be applied to a miRNA expression profile of the same at least 2 miRNAs as mentioned above (e.g. 10 miRNAs such as miRNAs according to SEQ ID NO: 1 to 10) of a subject (e.g. human or animal) to be diagnosed for a disease, preferably lung cancer and, thus, may then allow to discriminate whether the subject (e.g. human or animal) tested is healthy, i.e. not suffering from a disease, preferably lung cancer, or diseased, i.e. suffering from a disease, preferably lung cancer.
Additionally the algorithm may be trained to discriminate between more than 2 (e.g. 3, 4, 5 or more) clinical conditions from which at least one is a disease, preferably lung cancer.
Preferably, the reference and optionally the expression profile (data) of the miRNA(s) representative for a disease, preferably lung cancer is (are) stored in a database, e.g. an internet database, a centralized, and/or a decentralized database. It is preferred that the reference, e.g. mathematical function or algorithm, is comprised in a computer program, e.g. saved on a data carrier.
The above mentioned method is for diagnosing a disease, preferably lung cancer in a subject, e.g. a human or another mammal such as an animal. Preferably, the diagnosis comprises (i) determining the occurrence/presence of a disease, preferably lung cancer, (ii) monitoring the course of a disease, preferably lung cancer, (iii) staging of a disease, preferably lung cancer, (iv) measuring the response of a patient with a disease, preferably lung cancer to therapeutic intervention, and/or (v) segmentation of a subject suffering from a disease, preferably lung cancer.
Further, the above mentioned method is for prognosis of a disease, preferably lung cancer in a subject, a human or another mammal such as an animal. Preferably, the prognosis comprises (i) identifying of a subject who has a risk to develop a disease, preferably lung cancer, (ii) predicting/estimating the occurrence, preferably the severity of occurrence of a disease, preferably lung cancer, and/or (iii) predicting the response of a subject with a disease, preferably lung cancer to therapeutic intervention.
Further, in a preferred embodiment of the method of the present invention, for determining an expression profile of the set comprising at least two miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises a set of miRNAs listed in
For example, said set comprising 30 miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises a set of miRNAs listed in
Alternatively, said set comprising 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 miRNAs comprises a set of miRNAs listed in
For example, said set comprising 30 miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises a set of miRNAs listed in
Further, in another preferred embodiment of the method of the present invention, for determining an expression profile of the set comprising at least two miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises combinations of sets of miRNAs listed in
For example, said set comprising 30 miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises at least 2 sets of miRNAs listed in
For example, said set comprising 30 miRNAs representative for a disease, preferably lung cancer in a body fluid sample from a subject comprises a least 2 sets of miRNAs listed in
It is preferred in the method according to the first aspect of the invention that the set comprising at least two miRNAs representative for a disease is selected from the set of miRNAs listed in
It is preferred in the method according to the first aspect of the invention that the set comprising at least two miRNAs representative for lung cancer (NSCLC) is selected from the set of miRNAs listed in
In a second aspect, the invention relates to a set comprising polynucleotides for detecting a set comprising at least two miRNAs for diagnosing and/or prognosing of a disease in a body fluid sample from a subject.
It is preferred that the body fluid sample is a blood sample, particularly preferred it is a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred it is whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
Preferably, the set comprising at least two miRNAs is from the group consisting of SEQ ID NO: 1 to 222
Preferably the disease to be diagnosed or prognosed is lung cancer, particularly preferred the disease is non-small-cell lung carcinoma (NSCLC).
When diagnosing and/or prognosing lung cancer it is preferred that further nucleotide sequences of the miRNAs comprised in the set are selected from the group consisting of SEQ ID NO: 223 to 254, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is preferred that the set comprising at least two miRNAs is selected from the miRNAs listed in
It is preferred that
It is preferred that the polynucleotides of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 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, 35, 40 or more miRNAs, or comprising/consisting of 222 miRNAs and wherein the nucleotide sequences of said miRNAs are selected from the group consisting of SEQ ID NO: 1 to 222.
It is preferred that the polynucleotides of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, wherein the set comprising, miRNAs is selected from the set listed in
It is preferred that the polynucleotides of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 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, 35, 40 or more miRNAs, or comprising/consisting of 222 miRNAs and wherein the set of miRNAs comprises at least one of the sets listed in
For the body fluid sample (e.g. blood sample) analysis, it may be required that a set of polynucleotides (probes) capable of detecting a fixed defined set of miRNAs are attached to a solid support, bead, substrate, surface, platform, or matrix, e.g. biochip, which may be used for body fluid sample (e.g. blood sample) analysis. For example, if the fixed defined set of miRNAs for diagnosing a disease, preferably lung cancer comprises or consists of 20 miRNAs, polynucleotides capable of detecting these 20 miRNAs are attached to a solid support, substrate, surface, platform or matrix, e.g. biochip, in order to perform the diagnostic sample analysis.
Alternatively, it may be required that a set of chimeric polynucleotides (probes) capable of detecting a fixed defined set of miRNAs it contacted in solution with a sample containing miRNAs derived from a body fluid sample. The chimeric polynucleotide may comprise of a first sequence stretch that is complementary to a miRNA and a second sequence stretch that allows to pull down the chimeric polynucleotide-miRNA-duplexes to one or more solid supports (e.g. a set of beads for determining the set of miRNAs). For example, a set of 20 chimeric polynucleotides capable of detecting 20 miRNAs are contacted with sample containing miRNAs derived from a body fluid sample in order to form duplexes that can be pulled down to 20 different species of beads and detected thereon.
For example, the polynucleotides of the present invention are for detecting a set of 40 or 39 or 38 or 37 or 36 or 35 or 34 or 33 or 32 or 31 or 30 or 29 or 28 or 27 or 26 or 25 or 24 or 23 or 22 or 21 or 20 or 19 or 18 or 17 or 16 or 15 or 14 or 13 or 12 or 11 or 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 miRNAs wherein the set of miRNAs comprises at least one of the set of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 30 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 25 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 20 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 15 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 10 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
For example, the polynucleotides of the present invention are for detecting a set of 5 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
In a third aspect, the invention relates to the use of set of polynucleotides according to the second aspect of the invention for diagnosing and/or prognosing a disease, preferably lung cancer in a subject
In a fourth aspect, the invention relates to a set of at least two primer pairs for determining the expression level of a set of miRNAs in a body fluid sample of a subject suffering or suspected of suffering from a disease.
It is preferred that the body fluid sample is a blood sample, particularly preferred it is a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred it is whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
Preferably, the set comprising at least two miRNAs is from the group consisting of SEQ ID NO: 1 to 222
Preferably the disease to be diagnosed or prognosed is lung cancer, particularly preferred the disease is non-small-cell lung carcinoma (NSCLC).
When diagnosing and/or prognosing lung cancer it is preferred that further nucleotide sequences of the miRNAs comprised in the set are selected from the group consisting of SEQ ID NO: 223 to 254, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is preferred that the set of at least two primer pairs for determining the expression level of a set of miRNAs in a body fluid sample of a subject suffering or suspected of suffering from a disease are primer pairs that are specific for at least one miRNA listed in
It is preferred that the set of at least two primer pairs of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 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, 35, 36, 37, 38, 39, 40 or more miRNAs, or comprising/consisting of 222 miRNAs and wherein the nucleotide sequences of said miRNAs are selected from the group consisting of SEQ ID NO: 1 to 222.
It is preferred that the set of at least two primer pairs of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, wherein the set comprising, miRNAs is selected from the set listed in
It is preferred that the set of at least two primer pairs of the present invention are for detecting a set comprising, essentially consisting of, or consisting of at least 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, 35, 36, 37, 38, 39, 40 or more miRNAs, or comprising/consisting of 222 miRNAs and wherein the set of miRNAs comprises at least one of the sets listed in
For example, the set of at least two primer pairs of the present invention are for detecting a set of 40 or 39 or 38 or 37 or 36 or 35 or 34 or 33 or 32 or 31 or 30 or 29 or 28 or 27 or 26 or 25 or 24 or 23 or 22 or 21 or 20 or 19 or 18 or 17 or 16 or 15 or 14 or 13 or 12 or 11 or 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 miRNAs wherein the set of miRNAs comprises at least one of the set of miRNAs listed in
For example, the set of primer pairs of the present invention are for detecting a set of 30 miRNAs wherein the set of miRNAs comprises at least one of the sets of miRNAs listed in
Preferably, the said primer pairs may be used for amplifying cDNA transcripts of the set of miRNAs selected from the group consisting of SEQ ID 1 to 222. Furthermore, the said primer pairs may be used for amplifying cDNA transcripts of the set of miRNAs listed in
It is understood that the primer pairs for detecting a set of miRNAs may consist of specific and or non-specific primers. Additionally, the set of primer pairs may be complemented by other substances or reagents (e.g. buffers, enzymes, dye, labelled probes) known to the skilled in the art for conducting real time polymerase chain reaction (RT-PCR)
In a fifth aspect, the invention relates to the use of a set of primer pairs according to the fourth aspect of the invention for diagnosing and/or prognosing a disease, preferably lung cancer in a subject
In a sixth aspect, the invention relates to means for diagnosing and/or prognosing of a disease, preferably lung cancer in a body fluid sample of a subject.
Preferably, the invention relates to means for diagnosing and/or prognosing of a disease, preferably lung cancer in a body fluid sample of a subject comprising
It is preferred that the body fluid sample is a blood sample, particularly preferred it is a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred it is whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
Preferably, the set comprising at least two miRNAs is from the group consisting of SEQ ID NO: 1 to 222
Preferably the disease to be diagnosed or prognosed is lung cancer, particularly preferred the disease is non-small-cell lung carcinoma (NSCLC).
When diagnosing and/or prognosing lung cancer it is preferred that further nucleotide sequences of the miRNAs comprised in the set are selected from the group consisting of SEQ ID NO: 223 to 254, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is preferred that the set of at least two polynucleotides or the set of at least 2 primer pairs are for detecting a set comprising at least two miRNAs for diagnosing and/or prognosing of a disease, preferably lung cancer in a body fluid sample, e.g. blood sample, from a subject, e.g. patient, human or animal, wherein the set of miRNAs is selected from the miRNAs listed in
It is preferred that the set of at least two primer pairs for determining the expression level of a set of miRNAs in a body fluid sample of a subject suffering or suspected of suffering from a disease, preferably lung cancer are primer pairs that are specific for at least two miRNAs selected from the group consisting of SEQ ID 1 to 222.
It is preferred that the set of at least two primer pairs for determining the expression level of a set of miRNAs in a body fluid sample of a subject suffering or suspected of suffering from a disease, preferably lung cancer are primer pairs that are specific for at least one set of miRNAs listed in
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
The present invention provides means for diagnosing and/or prognosing of a disease, preferably lung cancer comprising a set comprising, essentially consisting of, or consisting of at least two polynucleotides (probes) according to the second aspect of the present invention, e.g. a polynucleotide for detecting a set comprising, essentially consisting of, or consisting of at least 2 polynucleotides, preferably comprising, essentially consisting of, or consisting of at least 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 or up to 222 or more polynucleotides for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, preferably comprising, essentially consisting of, or consisting of at least 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 or 40 or 222 miRNAs or all known miRNAs, wherein the nucleotide sequence of said miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
The means for diagnosing and/or prognosing of a disease, preferably lung cancer comprises, essentially consists of, or consists of a solid support, substrate, surface, platform or matrix comprising a set comprising, essentially consisting of, or consisting of at least two polynucleotides (probes) according to the second aspect of the present invention, e.g. a solid support, substrate, surface, platform or matrix comprising at least 2 polynucleotides, preferably comprising, essentially consisting of, or consisting of at least 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 or more polynucleotides, or comprising/consisting of 222 polynucleotides for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, preferably comprising, essentially consisting of, or consisting of at least 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 or more miRNAs, or comprising/consisting of 222 miRNAs, wherein the nucleotide sequence said miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto. Preferably, the above mentioned polynucleotide(s) is (are) attached or immobilized to the solid support, substrate, surface, platform or matrix. It is possible to include appropriate controls for non-specific hybridization on the solid support, substrate, surface, platform or matrix.
Additionally, the means for diagnosing and/or prognosing of a disease, preferably lung cancer comprises, essentially consists of, or consists of a solid support, substrate, surface, platform or matrix comprising a set comprising, essentially consisting of, or consisting of at least two polynucleotides (probes) according to the second aspect of the present invention, e.g. a solid support, substrate, surface, platform or matrix comprising at least 2 polynucleotides, preferably comprising, essentially consisting of, or consisting of at least 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 or more polynucleotides, or comprising/consisting of 222 polynucleotides for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, preferably comprising, essentially consisting of, or consisting of at least 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 or more miRNAs, or comprising/consisting of 222 miRNAs, wherein the set of miRNAs comprises at least one set of miRNAs listed in
It is particularly preferred that said means for diagnosing and/or prognosing of a disease, preferably lung cancer comprise, essentially consists of, or consists of a microarray/biochip comprising at least two polynucleotides according to the second aspect of the present invention.
It is also preferred that said means for diagnosing and/or prognosing of a disease, preferably lung cancer comprise, essentially consists of, or consists of a set of beads comprising a at least two polynucleotides according to the second aspect of the present invention. It is especially preferred that the beads are employed within a flow cytometer setup or a setup for analysing magnetic beads for diagnosing and/or prognosing of a disease, preferably lung cancer, e.g. in a LUMINEX system (www.luminexcorp.com)
Additionally, the present invention provides means for diagnosing and/or prognosing of a disease, preferably lung cancer comprising a set comprising, essentially consisting of, or consisting of at least two primer pairs according to the fourth aspect of the present invention, e.g. of at least 2 primer pairs, preferably comprising, essentially consisting of, or consisting of at least 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 or up to 222 or more primer pairs for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, preferably comprising, essentially consisting of, or consisting of at least 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 or 222 miRNAs or all known miRNAs, wherein the nucleotide sequence of said miRNA or the nucleotide sequences of said miRNAs is (are) preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
Also, the present invention provides means for diagnosing and/or prognosing of a disease, preferably lung cancer comprising a set comprising, essentially consisting of, or consisting of at least two primer pairs according to the fourth aspect of the present invention, e.g. of at least 2 primer pairs, preferably comprising, essentially consisting of, or consisting of at least 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 or up to 222 or more primer pairs for detecting a set comprising, essentially consisting of, or consisting of at least 2 miRNAs, preferably comprising, essentially consisting of, or consisting of at least 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 or 222 miRNAs or all known miRNAs, wherein the set of miRNAs comprises at least one set of miRNAs listed in
In a seventh aspect, the invention relates to a kit for diagnosing and/or prognosing of a disease, in a subject.
Preferably, the invention relates to a kit for diagnosing and/or prognosing of a disease comprising
The present invention provides a kit for diagnosing and/or prognosing of a disease, preferably lung cancer comprising
The present invention provides a kit for diagnosing and/or prognosing of a disease, preferably lung cancer comprising
Said means may comprise a set comprising, essentially consisting of, or consisting of at least two polynucleotides according to the second aspect of the present invention, a set of at least 2 primer pairs according to the fourth aspect of the invention; means according to the sixth aspect of the present invention; primers suitable to perform reverse transcriptase reaction and/or real time polymerase chain reaction such as quantitative polymerase chain reaction; and/or means for conducting next generation sequencing.
It is particularly preferred that said kit comprises
It is more particularly preferred that said kit comprises
Preferably, the above mentioned set comprising, essentially consisting of, or consisting of at least two polynucleotides are attached or immobilized to the solid support, substrate, surface, platform or matrix, e.g. to a microarray or to a set of beads.
Preferably, the above mentioned set comprising, essentially consisting of, or consisting of at least two polynucleotides is (are) attached or immobilized to microarray/biochip.
It is particularly preferred that said kit comprises
The primer as defined above may also be an oligo-dT primer, e.g. if the miRNA comprises a polyA tail (e.g. as a result of a miRNA elongation, for example, subsequent to RNA extraction) or a miRNA specific looped RT primer (Please amend/adapted if required).
It is also preferred that said kit comprises means for conducting next generation sequencing in order to determine an expression profile of a (single) miRNA or a set comprising, essentially consisting of, or consisting of at least 2 miRNAs representative for a disease, preferably lung cancer in a body fluid sample (e.g. blood sample) from a patient (e.g. human or animal), wherein the nucleotide sequence of said miRNA or the nucleotide sequences of said miRNAs is (are) preferably selected from the group consisting of SEQ ID NO: 1 to 222, a fragment thereof, and a sequence having at least 80% sequence identity thereto. Preferably, said kit further comprises means selected from the group consisting of: at least one body fluid sample, for example, tissue or blood sample, e.g. blood serum, blood plasma, or blood cells from a patient (e.g. human or animal), at least one sample of total RNA (or fractions thereof, e.g. miRNA) extracted from the body fluid sample (e.g. tissue or blood sample) of a patient (e.g. human or animal), and means to extract total RNA (or fractions thereof, e.g. miRNA) from a body fluid sample (e.g. blood sample).
The above mentioned kits further comprise at least one reference (ii). A comparison to said reference may allow for the diagnosis and/or prognosis of a disease, preferably lung cancer. Said reference may be the reference (e.g. reference expression profile (data)) of a healthy condition (i.e. not a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer), may be the reference (e.g. reference expression profile (data)) of a diseased condition (i.e. a disease, preferably lung cancer), or may be the reference (e.g. reference expression (data)) of at least two conditions from which at least one condition is a diseased condition (i.e. a disease, preferably lung cancer).
It is preferred that said reference is a reference expression profile (data) of at least one subject (e.g. human or animal), preferably the reference is an average expression profile (data) of at least 2 to 200 subjects, more preferably at least 10 to 150 subjects, and most preferably at least 20 to 100 subjects, with one known clinical condition which is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer, or which is not a disease, preferably lung cancer or not a specific form of a disease, preferably lung cancer (i.e. healthy/healthiness), wherein the reference expression profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs which expression profile is determined by the means of (i).
It is also preferred that said reference are (average) reference expression profiles (data) of at least two subjects, preferably of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects, with at least two known clinical conditions, preferably at least 2 to 5, more preferably at least 2 to 4 (i.e. at least 2, 3, 4, or 5) known clinical conditions, from which at least one is a disease, preferably lung cancer), wherein the reference expression profiles are the profiles of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs which expression profile is determined by the means of (i).
It is preferred that the reference is generated from expression profilies (data) obtained from 2 clinical conditions, which are a disease, preferably lung cancer and healthy control.
Preferably, (i) the (average) reference expression profile (data), which is provided with the kit, is determined in the same type of body fluid sample (e.g. blood and/or urine sample) and/or obtained from (control) subject(s) of the same species, gender and/or of similar age/stage of life, or (ii) the (average) reference expression profiles (data), which are provided with the kit, are determined in the same type of body fluid sample (e.g. blood and/or urine sample) and/or are obtained from (control) subject(s) of the same species, gender and/or of similar age/stage of life.
Said reference, preferably said (average) reference expression profile(s) (data) may be comprised in an information leaflet (e.g. for comparing tested single reference miRNA biomarkers with the expression profile data of a patient to be diagnosed) or saved on a data carrier (e.g. for comparing tested sets of miRNA biomarkers with the expression profile data of a patient to be diagnosed). Said reference, preferably said (average) reference expression profile(s) (data) may also be comprised in a computer program which is saved on a data carrier. The kit may alternatively comprise an access code which allows the access to a database, e.g. an internet database, a centralized or a decentralized database, where said reference, preferably said (average) reference expression profile(s) (data) is (are) comprised.
It is particularly preferred that the reference is an algorithm or mathematical function.
Preferably the algorithm or mathematical function is obtained from a reference expression profile (data) of at least one subject, preferably the algorithm or mathematical function is obtained from an average reference expression profile (data) of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects, i.e. of at least 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, or 40 subjects, with one known clinical condition which is a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer, or which is not a disease, preferably lung cancer or a specific form of a disease, preferably lung cancer (i.e. healthy/healthiness), wherein the reference expression profile is the profile of a single miRNA that has a nucleotide sequence that essentially corresponds (is essentially identical), preferably that corresponds (is identical), to the nucleotide sequence of the miRNA which expression profile is determined by the means of (i), or is the profile of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs which expression profile is determined by the means of (i).
It is also preferred that the algorithm or mathematical function is obtained from (average) reference expression profiles (data) of at least two subjects, preferably of at least 2 to 200 subjects, more preferably of at least 10 to 150 subjects, and most preferably of at least 20 to 100 subjects, i.e. of at least 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, or 40 subjects, with at least two known clinical conditions, preferably at least 2 to 5, more preferably at least 2 to 4 (i.e. at least 2, 3, 4, or 5) known clinical conditions, from which at least one is a disease, preferably lung cancer, wherein the reference expression profiles are the profiles of a single miRNA that has a nucleotide sequence that essentially corresponds (is essentially identical), preferably that corresponds (is identical), to the nucleotide sequence of the miRNA which expression profile is determined by the means of (i) or are the profiles of a set comprising at least two miRNAs that have nucleotide sequences that essentially correspond (are essentially identical), preferably that correspond (are identical), to the nucleotide sequences of the miRNAs which expression profile is determined by the means of (i).
It is preferred that the algorithm or mathematical function is obtained using a machine learning approach (see second aspect of the present invention).
Preferably, the algorithm or mathematical function is saved on a data carrier comprised in the kit or the computer program, wherein the algorithm or mathematical function is comprised, is saved on a data carrier comprised in the kit. Said kit may alternatively comprise an access code which allows the access to an internet page, where the algorithm or mathematical function is saved or where the computer program, wherein the algorithm or mathematical function is comprised, can be downloaded.
Preferably, the algorithm or mathematical function is saved on a data carrier or the algorithm or mathematical function is comprised in a computer program which is saved on a data carrier. Said kit may alternatively comprise an access code which allows the access to a database or an internet page, where the algorithm or mathematical function is comprised, or where a computer program comprising the algorithm or mathematical function can be downloaded.
More than one reference may be comprised in the kit, e.g. 2, 3, 4, 5, or more references. For example, the kit may comprise reference data, preferably (average) reference expression profile(s) (data), which may be comprised in an information leaflet or saved on a data carrier. In addition, the kit may comprise more than one algorithm or mathematical function, e.g. two algorithms or mathematical functions, e.g. one trained to discriminate between a healthy condition and a disease, preferably lung cancer and one trained to discriminate between specific forms of a disease, preferably lung cancer, e.g. comprised in a computer program, preferably stored on a data carrier.
In an eighth aspect, the invention relates to a set of miRNAs isolated from a body fluid sample from a subject for diagnosing and/or prognosing of a disease, wherein the miRNAs are selected from the group consisting of SEQ ID 1 to 222.
It is preferred that the body fluid sample is a blood sample, particularly preferred it is a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred it is whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the subject is a mammal including both a human and another mammal, e.g. an animal such as a mouse, a rat, a rabbit, or a monkey. It is particularly preferred that the subject is a human.
Preferably, the set comprising at least two miRNAs is from the group consisting of SEQ ID NO: 1 to 222
Preferably the disease to be diagnosed or prognosed is lung cancer, particularly preferred the disease is non-small-cell lung carcinoma (NSCLC).
When diagnosing and/or prognosing lung cancer it is preferred that further nucleotide sequences of the miRNAs comprised in the set are selected from the group consisting of SEQ ID NO: 223 to 254, a fragment thereof, and a sequence having at least 80% sequence identity thereto.
It is preferred that in the set of miRNAs isolated according to the eighth aspect of the invention at least one nucleotide sequences of the miRNAs comprised in the set is selected from the group consisting of SEQ ID NO: 1 to 12, a fragment thereof, and a sequence having at least 90% sequence identity thereto. It is particularly preferred that at least one nucleotide sequences of the miRNAs comprised in the set is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, a fragment thereof, and a sequence having at least 90% sequence identity thereto.
Preferably, the predetermined set comprising at least two miRNAs that are differentially regulated in blood samples from a disease patients as compared to healthy controls is selected from the miRNAs listed in
It is preferred that the predetermined set comprising at least two miRNAs that are differentially regulated in blood samples from a disease, preferably lung cancer patients as compared to healthy controls comprises at least one miRNA listed in
It is preferred in the set of miRNAs isolated according to the eighth aspect of the invention that the set comprising at least two miRNAs representative for a disease is selected from the set of miRNAs listed in
It is preferred in the set of miRNAs isolated according to the eighth aspect of the invention that the set comprising at least two miRNAs representative for lung cancer (NSCLC) is selected from the set of miRNAs listed in
In a ninth aspect, the invention relates to the use of a set of miRNAs according to the eighth aspect of the invention for diagnosing and/or prognosing of a disease, preferably lung cancer in a subject,
In a further aspect, the present invention relates to a method for diagnosing and/or prognosing of a disease, preferably lung cancer comprising the steps of:
The term “patient with an unknown clinical condition” refers to a patient (e.g. human or animal) which may suffer from a disease, preferably lung cancer (i.e. diseased patient) or may not suffer from a disease, preferably lung cancer (i.e. healthy patient). The patient (e.g. human or animal) to be diagnosed may further suffer from a specific type of a disease, preferably lung cancer. It is also possible to determine, whether the patient (e.g. human or animal) to be diagnosed will develop the above mentioned disease as the inventors of the present invention surprisingly found that miRNAs representative for a disease, preferably lung cancer are already present in the body fluid sample, e.g. blood sample, before a disease, preferably lung cancer occurs or during the early stage of a disease, preferably lung cancer. It should be noted that a patient that is diagnosed as being healthy, i.e. not suffering from a disease, preferably lung cancer, may possibly suffer from another disease not tested/known.
In a further aspect, the present invention relates to new nucleotide sequences for non-invasive diagnosis and/or prognosis of diseases. The nucleotide sequences for non-invasive diagnosis and/or prognosis of diseases are selected from the group consisting of SEQ ID NO: 1-222. The non-invasive diagnosis and/or prognosis of diseases according to the present invention is based on the analysis of nucleotide sequences selected from the group consisting of SEQ ID NO: 1-222 in a body fluid sample of a subject, preferably in a blood sample, particularly preferred in a whole blood, a blood cell, a PBMC, a serum, a plasma or a leukocyte or a leukocyte containing sample, more particularly preferred in whole blood sample containing at least red blood cells, platelets and granulocytes.
The present invention further relates to an isolated nucleic acid molecule selected from the group consisting of
The present invention further relates to an isolated nucleic acid molecule selected from the group consisting of
In still a further aspect of the invention relates to a nucleic acid or a set of nucleic acids for use in diagnosing and/or prognosing of a disease.
Preferably the nucleotide sequence of the nucleic acid for use in diagnosing and/or prognosing of a disease
Preferably at least one nucleotide sequence of the set of nucleic acids for use in diagnosing and/or prognosing of a disease
It is preferred that the diagnosing and/or prognosing using said nucleic acid or said set of nucleic acids is performed from a blood sample, preferably from a whole blood sample, more preferably from a whole blood sample containing at least red blood cells, platelets and granulocytes.
It is preferred that the disease to be diagnosed and/or prognosed using said nucleic acid or said set of nucleic acids is cancer, preferably lung cancer, more preferably non-small cell lung cancer.
In summary, the present invention is composed of the following items:
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.
Study Population
For the next-generation sequencing approach, we obtained whole blood samples from ten patients with NSCLC and ten healthy individuals (
For qRT-PCR we obtained lung cancer tissue from 16 different patients. We combined the RNA from those tissues to four pools, i.e., one squamous cell lung cancer pool, one adenocarcinoma pool, one large cell lung cancer pool, and one small cell lung cancer pool.
Considering the ethnic groups, all individuals were Caucasians with except of one Persian among the healthy blood donors. The enclosed lung cancer patients did not undergo any radio- or chemotherapy before blood drawing and tumor resection, all tumor patients were smokers or former smokers with 7 to 80 packyears.
Local ethics committee has approved the analysis of blood and tissue from patients and controls and participants have given their informed consent. We collected 2.5-5 ml whole blood in PAXgene™ Blood RNA tubes (PreAnalytiX) and stored the samples at −20° C. until extraction of total RNA. Further, we analyzed RNA isolated from 16 lung cancer tissue samples.
Isolation of Total RNA from Blood Cells and Tissue
Blood of patients has been extracted as previously described [1,2]. In brief, 2.5 to 5 ml blood was extracted in PAXgene Blood RNA tubes (BD, Franklin Lakes, N.J. USA) and centrifuged at 5000×g for 10 min at room temperature. The miRNeasy kit (Qiagen GmbH, Hilden) was used to isolate total RNA including miRNA from the resuspended pellet according to manufacturer's instructions. The eluted RNA was stored at −70° C.
For the isolation of RNA from tissue, samples were homogenized in 2 ml QIAzol lysis reagent and incubated for 5 min at RT. Then 200 μl chloroform were added, vortexed for 15 sec, and incubated for 2-3 min at RT. Subsequently, we followed the same protocol as applied for blood.
Library Preparation
1.5 μg of total RNA was enriched for the fraction of small RNAs (10-40 nt) using Ambion's flashPAGE Fractionator, followed by sodium acetate precipitation. SOLiD internal adapters were ligated using 100 ng enriched fraction. After ligation, small RNAs were transcribed into cDNA with Reverse Transcriptase. cDNA fragments between 60 and 80 nt (small RNAs+adaptors) were isolated from a 10% TBE Urea Gel (Novex-System, Invitrogen). RNA from gel slices was amplified with 15 PCR cycles using the same 5′-Primer for each sample and ten different 3′-Primers including the barcode sequences (SOLiD Multiplexing Barcoding Kit 01-16). A total of ten purified and barcoded DNA libraries was analyzed with a HS-DNA Chip in the Agilent Bioanalyzer 2100 and subsequently pooled in equimolar amounts.
Next Generation Sequencing
The pooled libraries were diluted to a concentration of 41 pg/μl. DNA was amplified monoclonally on magnetic beads in an emulsion PCR. Emulsions were broken with butanol and the remaining oil was washed off the templated double-stranded beads. DNA on the bead surface was denatured to allow hybridization of the enrichment beads to the single stranded DNA. Using a glycerol cushion the null beads can be separated from the templated beads. After centrifugation, the enriched magnetic beads were in the supernatant. The enrichment-beads were separated from the magnetic beads by denaturation. The 3′-end was enzymatically modified for deposition on the sequencing slide. 700 Million Beads were loaded onto a Full Slide and sequenced on a SOLiD 4 analyzer.
Mapping of Reads
Mapping of SOLiD sequencing reads against known miRNAs and the genome was done using the RNA2MAP tool (version 0.5) from Applied Biosystems (http://solidsoftwaretools.com/gf/project/rna2map/). To use the default parameters of this mapping pipeline, we first trimmed the reads to a size of 35 nt. To reduce the overhead of computation, we reduced the amount of reads per sample to those being unique in the sample. The RNA2MAP pipeline included three steps: 1) reads are filtered against tRNAs, rRNAs, and other repetitive elements; 2) the remaining reads are mapped against the predicted precursor sequences of miRNAs from miRBase (version 16 [7-9]); 3) the remaining reads are mapped against the human genome (hg19). The mapped genome reads served as input for the prediction of novel miRNAs with miRDeep [10]. The predicted novel miRNA precursor sequences were added to the precursor sequences of miRBase and step 2 of the RNA2MAP pipeline was repeated to retrieve the counts for both the known and novel predicted precursor sequences.
Prediction of Novel miRNAs
For the prediction of novel miRNAs, we used a probabilistic model of miRNA biogenesis in combination with the frequency of RNA reads along the secondary structure of the miRNA precursor as implemented in miRDeep [10]. Previously, we transformed the output of the alignments of RNA2MAP to the so-called ‘blastparsed’ format of miRDeep. To this end, we removed the sequencing adaptor, converted the colorspace mapping into bases, re-counted the mismatches, adjusted the alignment length, and computed a bitscore and an E-value as described previously [11]. The miRDeep pipeline itself was run with default parameters using Randfold (v 2.0, [12]) and a fasta file containing the mature miRNA sequences from miRBase v16 (without human sequences) to improve accuracy and sensitivity. To reduce the number of false positive predictions, we ran 100 permutation tests and excluded a predicted novel miRNA if found in any of the permutation runs. The remaining putative novel miRNAs (p-value <0.01) were mapped with BLAST (v 2.2.24, [13]) against known ncRNA and miRNA sequences from diverse sources (miRBase v16, snoRNA-LBME-db [14], ncRNAs from Ensembl “Homo_sapiens. GRCh37.59.ncrna.fa” ((ftp://ftp.ensembl.org/pub/release-59/fasta/homo_sapiens/ncrna/). NONCODE v2.0, [15]). We excluded sequences that aligned with more than 90% of their length (allowing 1 mismatch) to any of the ncRNA sequences.
Distribution of miRNA Reads Across the miRNA Precursors
Since we performed a size selection we do not intent to measure the expression level of the miRNA-precursor but of the mature miRNAs. The mapping of mature miRNA reads to the respective precursor sequence however offers the option to understand how the mature miRNA reads distribute along the precursor. To consider the distribution of reads mapping to a miRNA precursor, we computed for each precursor separately the coverage of each base position for lung cancer samples and controls. Likewise, we also computed for each base position of each precursor a significance value using Wilcoxon Mann-Whitney (WMW) test.
Downstream Analysis
To further evaluate the NGS miRNA profiles, we carried out statistical computations using R [16]. Shapiro-Wilk test has been applied to determine whether miRNA counts across all samples are normally distributed. To normalize samples standard quantile normalization has been applied to make the different sequencing runs comparable to each other. Expression of a miRNA i in a sample j has been measured as the normalized read count of this miRNAs in the respective sample. Grubbs test has been carried out for detecting outliers. Non-parametric WMW test has been performed for detecting differentially regulated miRNAs. To further assess the validity of the signature we have carried out non-parametric permutation tests. Here, the class labels have been randomly shuffled 100 times and the same analyses as for the original class labels have been carried out. A p-value was computed as the fraction of random runs with a likewise significant result as the original computations.
In addition to WMW analysis, we performed an analysis considering the total length of a miRNA precursor to identify possible novel miRNAs that derive from this precursor. In detail, we computed for each precursor m and each base i the WMW significance value for the respective position in the precursor at position i, testing the hypothesis that read counts of miRNA m at position i are significantly higher for lung cancer samples as compared for normal controls. For each miRNA precursor, we then counted the number of bases with WMW significance values <0.05. Furthermore, the area under the receiver operator characteristics (AUC) curve has been computed for each miRNA. Cluster analysis has been done using the ‘hclust’ package.
For computing targets of deregulated miRNAs, the miRANDA algorithm has been applied and only miRNA-mRNA relations with p-values <0.0001 have been considered [17]. To carry out gene set enrichment of target genes, we used GeneTrail and carried out a so-called over representation analysis [18, 19].
| Number | Date | Country | Kind |
|---|---|---|---|
| 11184241.5 | Oct 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2012/069725 | 10/5/2012 | WO | 00 |
