Patent Application
20100261617
The present invention refers to a method of monitoring, in particular prognosing and risk stratification of prostate cancer by the means of expression profiling.
Prostate cancer (PCA) is the most frequent tumor type in males and a major cause of death due to malignancy. It represents the cause of 6% of cancer deaths in men. Based on some estimates, 80% of men over the age of 80 suffer of some form of prostate disease (e.g. cancer, Benign Prostatic Hypertrophy, prostatitis, etc.).
Current methods for prostate cancer screening include a blood test for prostate specific antigen (PSA), digital rectal examination (DRE), and transrectal ultrasound (TRUS), whereas the latter is mainly used to image the prostate and to aid in guided needle biopsy. The widespread use of the prostate specific antigen (PSA) for the detection of PCA has resulted in an increasing number of men diagnosed with organ-confined, low Gleason-score PCA, which are potentially curable. However, the high sensitivity of the PSA test is accompanied by a low specificity, causing many patients suffering from unnecessary biopsy taking. Men with normal prostate pathology generally have a PSA level in blood below 4 ng/ml. PSA levels between 4 ng/ml and 10 ng/ml (‘grey zone’) have a 25% chance of having prostate cancer. As a consequence, in 75% of the cases, men with an abnormal DRE and a PSA in this grey zone have a negative, or a seemingly unnecessary biopsy.
Thus, the object of the present invention is to find new molecular markers to improve early diagnosis, prediction of progression, and therapy of PCA. Because of their clinical importance, a better understanding of the molecular mechanisms of these mostly small tumors is essential in order to identify novel diagnostic and prognostic biomarkers for a tailored clinical management in the individual patient. Furthermore, in prostate cancer, heterogeneity is a common phenomenon which includes different histological grades within the same tumor focus and different genotypes among phenotypically similar foci in a single tumor. To better understand the molecular mechanisms of prostate cancer, it is essential to correlate gene expression with a specific cell type. Therefore, sub-populations of cells have to be selected to perform comparable molecular profiling experiments with the highest grade of information and comparability.
The WO2006/091776 discloses the identity of 41 genes the expression at the transcriptional and translational levels of which has been found to correlate with cancer progression. Furthermore, Schlomm et al. (International Journal of Oncology, 2005, Vol. 27, pages 713-720) have identified a set of genes which are shown to be either up- or down-regulated in prostate-derived tumor tissue as compared to healthy tissue. However, use of these genes as alternative or supplemental diagnostically or otherwise clinically useful markers in a commercial setting has not been enabled.
Thus, the problem of the present invention resides in the lack of a reliable method to clearly monitor the presence of a prostate tumor and to perform molecular classification of prostata cancer stages, which allows for an early determination of the potential aggressiveness of the tumor, the respective prognosis, and, thus, an optimal mode of therapy.
The problem is solved by the present invention, which provides
a method for monitoring the presence and/or progression of prostate tumor in an individual, the method comprising:
According to the present invention, the term “monitoring” encompasses i) diagnosing whether an individual is suffering from a prostate disease, in particular prostate cancer; ii) prognosing and assessing the risk of the disease progression by determining up to which stage the tumor has already progressed; and iii) selecting specified treatments based on the prognosis provided to the patient through determining the expression levels of the markers of the present invention. Thus, the method of the present invention provides, through monitoring of the expression levels suitable means to evaluate which patients may benefit from a more aggressive treatment, and which patients could be spared from unnecessary treatments.
The term “prostate tumor progression” refers to the general classification of prostate tumor stages, which is well-known to the skilled artisan. In brief, the most commonly used staging method is the Tumour, Nodes, Metastasis (TNM) system, which recognises four stages of local tumour growth, from T1 (incidental) to T4 (invasion of neighbouring organs). Each stage describes the state of pathological development of the tumour. T1 represents an ‘incidental’ state, where the tumour is detected by chance following transurethral resection or by biopsy following PSA testing. At this stage, the tumour will be undetectable by palpation (DRE) or ultrasonography, but may be diagnosed by the method of the present invention. T4 represents advanced disease, where the tumour has invaded neighbouring organs. The nodal stage (N0-N1) and the metastatic stage (M0-M1C) reflect the clinical spread of the disease to lymph nodes and distant sites (metastasis), respectively. Grading systems can assess the degree of cell anaplasia (variation in size, shape and staining properties) and differentiation (how well differentiated the cells are) in the tumour. The Gleason grading system is based on the extent to which the tumour cells are arranged into recognisably glandular structures and the level of cell differentiation. The Gleason system identifies more than five levels of increasing disease aggressiveness, with Grade 1 being the least aggressive and over Grade 5 being the most aggressive cancer.
According to the present invention, a “sample” means any biological material containing genetic information in the form of nucleic acids or proteins obtainable or obtained from an individual. The sample includes e.g. tissue samples, cell samples, bone marrow and/or body fluids such as blood, serum, urin, saliva, semen. Preferably, the sample is tissue or cell samples. The person skilled in the art is aware of methods, how to isolate nucleic acids and proteins from a sample. A general method for isolating and preparing nucleic acids from a sample is outlined in Example 1.
According to the present invention, the term “expression” refers to the process by which mRNA or a polypeptide is produced based on the nucleic acid sequence of a gene, i.e. “expression” also includes the formation of mRNA upon transcription. In accordance with the present invention, the term “determining the expression level” preferably refers to the determination of the level of expression, namely of the markers.
Generally, “marker” refers to any genetically controlled difference which can be used in the genetic analysis of a test versus a control sample, for the purpose of assigning the sample to a defined genotype or phenotype. As used herein, “markers” refer to genes which are differentially expressed in, e.g., cancer patients with highly progressed prostate tumor stages accompanied with bad survival prognosis. The markers can be defined by their gene symbol name, their encoded protein name, their transcript identification number (cluster identification number), the data base accession number, public accession number or GenBank (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) identifier or chromosomal location, UniGene accession number and cluster type, LocusLink accession number (see Examples and Tables).
Generally, the expression level of a marker is determined by the determination of the expression of its corresponding “polynucleotide” or “polypeptide” amounts as described hereinafter.
According to the present invention, the term “polynucleotide” refers, generally, to a DNA, in particular cDNA, or RNA, in particular a cRNA, or a portion thereof or a polypeptide or a portion thereof. In the case of RNA (or cDNA), the polynucleotide is formed upon transcription of a nucleotide sequence which is capable of expression. The polynucleotide fragments refer to fragments preferably of between at least 8, such as 10, 12, 15 or 18 nucleotides and at least 50, such as 60, 80, 100, 200 or 300 nucleotides in length, or a complementary sequence thereto, representing a consecutive stretch of nucleotides of a gene, cDNA or mRNA. In other terms, polynucleotides include also any fragment (or complementary sequence thereto) of a sequence derived from any of the markers defined above as long as these fragments unambiguously identify the marker.
The determination of the expression level may be effected at the transcriptional or translational level, i.e. at the level of mRNA or at the protein level. Protein fragments such as peptides or “polypeptides” advantageously comprise between at least 6 and at least 25, such as 30, 40, 80, 100 or 200 consecutive amino acids representative of the corresponding full length protein. Six amino acids are generally recognized as the lowest peptidic stretch giving rise to a linear epitope recognized by an antibody, fragment or derivative thereof. Alternatively, the proteins or fragments thereof may be analysed using nucleic acid molecules specifically binding to three-dimensional structures (aptamers).
Depending on the nature of the marker, i.e. whether it is in form of a polynucleotide or polypeptide, the determination of the expression levels may be effected by a variety of methods. For determining and detecting the expression level, it is preferred in the present invention that the marker is labelled.
The labelling of the marker, i.e. the polynucleotide or its corresponding polypeptide can occur by a variety of methods known to the skilled artisan. The label can be fluorescent, chemiluminescent, bioluminescent, radioactive (such as 3H or 32P). The labelling compound can be any labelling compound being suitable for the labelling of polynucleotides and/or polypeptides. Examples include fluorescent dyes, such as fluorescein, dichlorofluorescein, hexachlorofluorescein, BODIPY variants, ROX, tetramethylrhodamin, rhodamin X, Cyanine-2, Cyanine-3, Cyanine-5, Cyanine-7, IRD40, FluorX, Oregon Green, Alexa variants (available e.g. from Molecular Probes or Amersham Biosciences) and the like, biotin or biotinylated nucleotides, digoxigenin, radioisotopes, antibodies, enzymes and receptors. Depending on the type of labelling, the detection is done via fluorescence measurements, conjugation to streptavidin and/or avidin, antigen-antibody- and/or antibody-antibody-interactions, radioactivity measurements, as well as catalytic and/or receptor/ligand interactions. Suitable methods include the direct labelling (incorporation) method, the amino-modified (amino-allyl) nucleotide method (available e.g. from Ambion), and the primer tagging method (DNA dendrimer labelling, as kit available e.g. from Genisphere). Particularly preferred for the present invention is the use of biotin or biotinylated nucleotides for labelling, with the latter being directly incorporated into, e.g. the cRNA polynucleotide by in vitro transcription.
If the polynucleotide is mRNA, cDNA may be prepared into which a detectable label, as exemplified above, is incorporated. Said detectably labelled cDNA, in single-stranded form, may then be hybridised, preferably under stringent or highly stringent conditions to a panel of single-stranded oligonucleotides representing different genes and affixed to a solid support such as a chip. Upon applying appropriate washing steps, those cDNAs will be detected or quantitatively detected that have a counterpart in the oligonucleotide panel. Various advantageous embodiments of this general method are feasible. For example, the mRNA or the cDNA may be amplified e.g. by polymerase chain reaction, wherein it is preferable, for quantitative assessments, that the number of amplified copies corresponds relative to further amplified mRNAs or cDNAs to the number of mRNAs originally present in the cell. In a preferred embodiment of the present invention, the cDNAs are transcribed into cRNAs prior to the hybridisation step wherein only in the transcription step a label is incorporated into the nucleic acid and wherein the cRNA is employed for hybridisation. Alternatively, the label may be attached subsequent to the transcription step.
Similarly, proteins from a cell or tissue under investigation may be contacted with a panel of aptamers or of antibodies or fragments or derivatives thereof. The antibodies etc. may be affixed to a solid support such as a chip. Binding of proteins indicative of disease or non-disease may be verified by binding to a detectably labelled secondary antibody or aptamer. For the labelling of antibodies, it is referred to Harlow and Lane, “Antibodies, a laboratory manual”, CSH Press, 1988, Cold Spring Harbor. Specifically, a minimum set of proteins necessary for monitoring the presence and/or progression of prostate tumor may be selected for creation of a protein array system to make diagnosis on a protein lysate of a diagnostic tissue sample directly. Protein Array Systems for the detection of specific protein expression profiles already are available (for example: Bio-Plex, BIORAD, München, Germany). For this application preferably antibodies against the proteins have to be produced and immobilized on a platform e.g. glasslides or microtiterplates. The immobilized antibodies can be labelled with a reactant specific for the certain target proteins as discussed above. The reactants can include enzyme substrates, DNA, receptors, antigens or antibodies to create for example a capture sandwich immunoassay.
For reliably monitoring the presence and/or progression of prostate tumor it is useful that the expression of more than one of the above defined markers is determined. As a criterion for the choice of markers, the statistical significance of markers as expressed in q or p values based on the concept of the false discovery rate is determined. In doing so, a measure of statistical significance called the q value is associated with each tested feature. The q value is similar to the p value, except it is a measure of significance in terms of the false discovery rate rather than the false positive rate (Storey J D and Tibshirani R. Proc. Natl. Acad. Sci., 2003, Vol. 100:9440-5.
Of the above defined markers, the expression level of at least two, preferably of at least ten, more preferably of at least 25, most preferably of 39 of the markers in Table 1 is determined.
In another preferred embodiment, the expression level of at least 2, of at least 5, of at least 10 out of the markers having the SEQ ID NOs: 1-12, 1-20, 1-39, of Table 1 are measured.
The level of the expression of the “marker”, i.e. the expression of the polynucleotide is indicative for the presence of prostate tumor of a cell or an organism. The level of expression of a marker or group of markers is measured and is compared with the level of expression of the same marker or the same group of markers from other cells or samples. The comparison may be effected in an actual experiment or in silico. When the expression level also referred to as expression pattern or expression signature (expression profile) is measurably different, there is according to the invention a meaningful (i.e. statistically significant) difference in the level of expression. Preferably the difference at least is 5%, 10% or 20%, more preferred at least 50% or may even be as high as 75% or 100%. More preferred the difference in the level of expression is at least 200%, i.e. two fold, at least 500%, i.e. five fold, or at least 1000%, i.e. 10 fold.
Accordingly, the expression level of markers expressed lower in a disease sample than in a healthy, normal sample is at least 5%, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold lower, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold lower in the disease sample. On the other hand, the expression level of markers expressed higher in a disease sample than in a healthy, normal sample is at least 5%, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold higher, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold higher in the disease sample.
For the method of the present invention it is preferred if the polynucleotide the expression level of which is determined is in form of a transcribed polynucleotide. A particularly preferred transcribed polynucleotide is an mRNA, a cDNA and/or a cRNA, with the latter being preferred. Transcribed polynucleotides are isolated from a sample, reverse transcribed and/or amplified, and labelled, by employing methods well-known the person skilled in the art (see Examples). In a preferred embodiment of the methods according to the invention, the step of determining the expression profile further comprises amplifying the transcribed polynucleotide.
In order to determine the expression level of the transcribed polynucleotide by the method of the present invention, it is preferred that the method comprises hybridizing the transcribed polynucleotide to a complementary polynucleotide, or a portion thereof, under stringent hybridization conditions, as described hereinafter.
The term “hybridizing” means hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook, J., et al., in “Molecular Cloning: A Laboratory Manual” (1989), Eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. and the further definitions provided above. Such conditions are, for example, hybridization in 6×SSC, pH 7.0/0.1% SDS at about 40 to 45°, preferably 42° C. for 16-23 hours, followed by a washing step with 2×SSC/0.1% SDS at 30 to 50° C. In order to select the stringency, the salt concentration in the washing step can for example be chosen between 2× down to 0.5×SSC/0.1% SDS at room temperature for low and medium stringency and 0.2× down to 0.05×SSC/0.1% SDS at 30 to 50° C. for high stringency. In addition, the temperature of the washing step can be varied between room temperature, ca. 22° C., for low stringency, and 65° C. to 70° C. for high stringency. Preferably the washing temperature is 36° C.
Also contemplated are polynucleotides that hybridize at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation, preferably of formamide concentration (lower percentages of formamide result in lowered stringency), salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37° C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 mg/ml salmon sperm blocking DNA, followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
“Complementary” and “complementarity”, respectively, can be described by the percentage, i.e. proportion, of nucleotides which can form base pairs between two polynucleotide strands or within a specific region or domain of the two strands. Generally, complementary nucleotides are, according to the base pairing rules, adenine and thymine (or adenine and uracil), and cytosine and guanine. Complementarity may be partial, in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be a complete or total complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has effects on the efficiency and strength of hybridization between nucleic acid strands.
Two nucleic acid strands are considered to be 100% complementary to each other over a defined length if in a defined region all adenines of a first strand can pair with a thymine (or an uracil) of a second strand, all guanines of a first strand can pair with a cytosine of a second strand, all thymine (or uracils) of a first strand can pair with an adenine of a second strand, and all cytosines of a first strand can pair with a guanine of a second strand, and vice versa. According to the present invention, the degree of complementarity is determined over a stretch of 20, preferably 25, nucleotides, i.e. a 60% complementarity means that within a region of 20 nucleotides of two nucleic acid strands 12 nucleotides of the first strand can base pair with 12 nucleotides of the second strand according to the above ruling, either as a stretch of 12 contiguous nucleotides or interspersed by non-pairing nucleotides, when the two strands are attached to each other over said region of 20 nucleotides. The degree of complementarity can range from at least about 50% to full, i.e. 100% complementarity. Two single nucleic acid strands are said to be “substantially complementary” when they are at least about 80% complementary, preferably about 90% or higher. For carrying out the method of the present invention substantial complementarity is preferred.
Preferred methods for detection and quantification of the amount of polynucleotides, i.e. for the methods according to the invention allowing the determination of the level of expression of a marker, are those described by Sambrook et al. (1989) or real time methods known in the art as the TaqMan® method disclosed in WO92/02638 and the corresponding U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375, U.S. Pat. No. 5,487,972. This method exploits the exonuclease activity of a polymerase to generate a signal. In detail, the (at least one) target nucleic acid component is detected by a process comprising contacting the sample with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid component and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid component sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3′-end of the first oligonucleotide is adjacent to the 5′-end of the labeled oligonucleotide. Then this mixture is treated with a template-dependent nucleic acid polymerase having a 5′ to 3′ nuclease activity under conditions sufficient to permit the 5′ to 3′ nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments. The signal generated by the hydrolysis of the labeled oligonucleotide is detected and/or measured. TaqMan® technology eliminates the need for a solid phase bound reaction complex to be formed and made detectable. Other methods include e.g. fluorescence resonance energy transfer between two adjacently hybridized probes as used in the LightCycler® format described in U.S. Pat. No. 6,174,670.
A preferred protocol if the marker, i.e. the polynucleotide, is in form of a transcribed nucleotide, is described in Examples 1 and 2, where total RNA is isolated, cDNA is synthesized and a fluorescent dye is incorporated during the reverse transcription reaction. The purified cDNA is applied to high-density cDNA arrays. The hybridized cDNA is detected according to the methods described in Example 2. The arrays can be produced by photolithography or other methods known to experts skilled in the art e.g. from U.S. Pat. No. 5,445,934, U.S. Pat. No. 5,744,305, U.S. Pat. No. 5,700,637, U.S. Pat. No. 5,945,334 and EP 0 619 321 or EP 0 373 203, or as described hereinafter in greater detail.
In another embodiment of the present invention, the marker is in form of a polypeptide. In another preferred embodiment, the expression level of the polynucleotides or polypeptides is detected using a compound which specifically binds to the polynucleotide of the polypeptide of the present invention.
As used herein, “specifically binding” means that the compound is capable of discriminating between two or more polynucleotides or polypeptides, i.e. it binds to the desired polynucleotide or polypeptide, but essentially does not bind unspecifically to a different polynucleotide or polypeptide.
The compound can be an antibody, or a fragment thereof, an enzyme, a so-called small molecule compound, a protein-scaffold, preferably an anticalin. In a preferred embodiment, the compound specifically binding to the polynucleotide or polypeptide is an antibody, or a fragment thereof.
As used herein, an “antibody” comprises monoclonal antibodies as first described by Köhler and Milstein in Nature 278 (1975), 495-497 as well as polyclonal antibodies, i.e. entibodies contained in a polyclonal antiserum. Monoclonal antibodies include those produced by transgenic mice. Fragments of antibodies include F(ab′)2, Fab and Fv fragments. Derivatives of antibodies include scFvs, chimeric and humanized antibodies. See, for example Harlow and Lane, loc. cit. For the detection of polypeptides using antibodies or fragments thereof, the person skilled in the art is aware of a variety of methods, all of which are included in the present invention. Examples include immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA), Enzyme-linked immuno sorbent assay (RIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA). For detection, it is desirable if the antibody is labelled by one of the labelling compounds and methods described supra.
In another preferred embodiment of the present invention, the method for monitoring presence and/or progression of prostate tumor is carried out on an array.
In general, an “array” or “microarray” refers to a linear or two- or three dimensional arrangement of preferably discrete nucleic acid or polypeptide probes which comprises an intentionally created collection of nucleic acid or polypeptide probes of any length spotted onto a substrate/solid support. The person skilled in the art knows a collection of nucleic acids or polypeptide spotted onto a substrate/solid support also under the term “array”. As known to the person skilled in the art, a microarray usually refers to a miniaturised array arrangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules referring to different or the same genes per cm2. Furthermore, where appropriate an array can be referred to as “gene chip”. The array itself can have different formats, e.g. libraries of soluble probes or libraries of probes tethered to resin beads, silica chips, or other solid supports.
The process of array fabrication is well-known to the person skilled in the art. In the following, the process for preparing a nucleic acid array is described. Commonly, the process comprises preparing a glass (or other) slide (e.g. chemical treatment of the glass to enhance binding of the nucleic acid probes to the glass surface), obtaining DNA sequences representing genes of a genome of interest, and spotting sequences these sequences of interest onto glass slide. Sequences of interest can be obtained via creating a cDNA library from an mRNA source or by using publicly available databases, such as GeneBank, to annotate the sequence information of custom cDNA libraries or to identify cDNA clones from previously prepared libraries. Generally, it is recommendable to amplify obtained sequences by PCR in order to have sufficient amounts of DNA to print on the array. The liquid containing the amplified probes can be deposited on the array by using a set of microspotting pins. Ideally, the amount deposited should be uniform. The process can further include UV-crosslinking in order to enhance immobilization of the probes on the array.
The array can also be a high density oligonucleotide (oligo) array using a light-directed chemical synthesis process, employing the so-called photolithography technology. Unlike common cDNA arrays, oligo arrays use a single-dye technology. Given the sequence information of the markers, the sequence can be synthesized directly onto the array, thus, bypassing the need for physical intermediates, such as PCR products, required for making cDNA arrays. For this purpose, the marker, or partial sequences thereof, can be represented by 14 to 20 features, preferably by less than 14 features, more preferably less than 10 features, even more preferably by 6 features or less, with each feature being a short sequence of nucleotides (oligonucleotide), which is a perfect match (PM) to a segment of the respective gene. The PM oligonucleotide are paired with mismatch (MM) oligonucleotides which have a single mismatch at the central base of the nucleotide and are used as “controls”. The chip exposure sites are defined by masks and are deprotected by the use of light, followed by a chemical coupling step resulting in the synthesis of one nucleotide. The masking, light deprotection, and coupling process can then be repeated to synthesize the next nucleotide, until the nucleotide chain is of the specified length.
Advantageously, the method of the present invention is carried out in a robotics system including robotic plating and a robotic liquid transfer system, e.g. using microfluidics, i.e. channelled structured.
A particular preferred method according to the present invention is as follows:
1. Obtaining a sample, e.g. tissue samples, from a patient having prostate cancer
2. Extracting RNA, preferably mRNA, from the sample
3. Reverse transcribing the RNA into cDNA
4. Hybridizing the cDNA on standard microarrays
5. Determining hybridization
In another embodiment, the present invention is directed to the use of at least one marker selected from the markers comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and/or 39, for the manufacturing of a diagnostic for monitoring presence and/or progression of prostate tumor.
The use of the present invention is particularly advantageous for monitoring tumor progression in an individual suffering from a prostate disease. Preferred prostate diseases according to the present invention include prostatitis, benign prostatic hyperplasia, localized prostate cancer, hormone naïve metastatic prostate cancer, hormone refractory metastatic prostate cancer, or metastatic small cell prostate cancer.
In a preferred embodiment of the invention, the use is particularly advantageous for monitoring tumor progression in an individual suffering from prostate cancer.
The use of said markers for monitoring presence and/or progression of prostate tumor, preferably based on microarray technology, offers the following advantages: (1) more rapid and more precise diagnosis, (2) easy to use in laboratories without specialized experience, and (3) abolishes the requirement for analyzing viable cells for chromosome analysis.
Accordingly, the present invention refers to a diagnostic kit containing at least one marker selected from the markers comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and/or 39, for monitoring the presence and/or progression of prostate tumor, in combination with suitable auxiliaries. Suitable auxiliaries, as used herein, include buffers, enzymes, labelling compounds, and the like. In a preferred embodiment, the marker contained in the kit is a nucleic acid molecule which is capable of hybridizing to the mRNA corresponding to at least one marker of the present invention. Preferably, the at least one nucleic acid molecule is attached to a solid support, e.g. a polystyrene microtiter dish, nitrocellulose membrane, glass surface or to non-immobilized particles in solution.
In another preferred embodiment, the diagnostic kit contains at least one reference for a disease and/or a healthy, normal sample. As used herein, the reference can be a biological sample or a data base.
In another embodiment, the present invention is directed to an apparatus for monitoring the presence and/or progression of prostate tumor in a sample, the apparatus containing a reference data base obtainable by
The values obtained from the determination are saved and maintained in a recallable format, in order to be used for comparison with an unknown sample. In brief, expression values of more than one marker are determined for the sample to be tested, comprising: receiving the gene expression values for more than one marker in the sample to be tested; means for providing a model generated by a supervised learning algorithm based on a dataset of expression values from known samples; comparing the gene expression values of the sample to that of the model, to thereby produce a classification of the sample; and providing an output indication of the classification. Thus, the apparatus can include: a source of expression values of more than one marker in the sample; means for providing a model generated by a trained algorithm based on a dataset of expression values from known biological samples; a processor routine executed by a digital processor, coupled to receive the expression values from the source, the processor routine determining classification of the sample by comparing the expression values of the sample to the model; and an output assembly, coupled to the digital processor, for providing an indication of the classification of the sample
The apparatus of the present invention containing a desired reference data base can be used in a way such that an unknown sample is, first, subjected to gene expression profiling, e.g. by microarray analysis in a manner as described supra or in the art, and the expression level data obtained by the analysis are, second, fed into the apparatus and compared with the data of the reference data base obtainable by the above method. The classifying of the gene expression profile whether it is derived from a disease or healthy individual occurs by means of a machine learning algorithm.
According to the present invention, the “machine learning algorithm” is a computational-based prediction methodology, also known to the person skilled in the art as “classifier”, employed for characterizing a gene expression profile. The signals corresponding to a certain expression level which are obtained by the microarray hybridization are subjected to the algorithm in order to classify the expression profile. Supervised learning involves “training” a classifier to recognize the distinctions among classes and then “testing” the accuracy of the classifier on an independent test set. For new, unknown sample the classifier shall predict into which class—disease or healthy—the sample belongs.
Preferably, the machine learning algorithm is selected from the group consisting of Weighted Voting, K-Nearest Neighbors, Decision Tree Induction, Support Vector Machines (SVM) such as polynomial kernel and Gaussian Radial Basis Function-kernel SVM models, and Feed-Forward Neural Networks. Most preferably, the machine learning algorithm is K-Nearest Neighbors.
In a preferred embodiment, the reference data base is backed up on a computational data memory chip which can be inserted in as well as removed from the apparatus of the present invention, e.g. like an interchangeable module, in order to use another data memory chip containing a different reference data base.
The apparatus suitably contains a device for entering the expression level of the data, for example a control panel such as a keyboard. The results, whether and how the data of the unknown sample fit into the reference data base can be made visible on a provided monitor or display screen and, if desired, printed out on an incorporated of connected printer.
Alternatively, the apparatus of the present invention is equipped with particular appliances suitable for detecting and measuring the expression profile data and, subsequently, proceeding with the comparison with the reference data base. In this embodiment, the apparatus of the present invention can contain a gripper arm and/or a tray which takes up the microarray containing the hybridized nucleic acids.
In another embodiment, the present invention refers to a reference data base for monitoring the presence and/or progression of prostate tumor in an individual obtainable by comprising
Preferably, the reference data bank is backed up and/or contained in a computational memory data chip.
The invention is further illustrated and exemplified by the following examples, without limiting the scope of the invention:
Radical prostatectomy specimens are obtained from at least 20 patients. After removal, the prostate is macroscopically inspected. If a tumor is not visible or palpable, a single cut is made through the mid portion of the lateral surface of the gland from the basal to the apical section. Samples are taken with a 6 mm punch biopsy instrument (Biopsy Punch, Stiefel, Wächtersburg, Germany) from areas that are suspected to contain tumor foci based on information obtained from the preoperative systematic 10-location biopsies. Each individual sample is immediately stored in a kryo-tube filled with 1.5 ml RNAlater (Qiagen, Hilden, Germany). After storage overnight at ambient temperature, the specimens are transferred to a −20° C. freezer. In the case of a palpable or visible tumor, the same procedure is performed in the suspected tumor area. Non-cancerous tissue is obtained by the same procedure from areas assumed to be tumor-free. After finishing all tissue-collecting procedures, the sliced surfaces are painted with indelible yellow ink (Wak Chemie, Steinbach, Germany). All materials and solutions are either purchased in RNase-free state or treated to deactivate RNases. Ethanol dilutions are made with RNase-free water. First, in the cryo-tubes, the specimens are thawed at room temperature. To elute most of the RNAlater from the tissue, two subsequent washing steps of 5 min each in 10 ml precooled (0° C.) sterile PBS-buffer (PBS Dulbecco's, Invitrogen, Karlsruhe, Germany) are performed in an ice bath. These washings strongly facilitated downstream procedures, e.g. cryo cutting and laser microdissection as well as microscopic visualization, which are often influenced unfavorably by RNAlater. The washed specimens are directly applied to the holder of the cryo-microtome in a drop of Tissue-Tek® (OCT) at the optimal cutting temperature of −25° C. After thorough freezing, cryo sections are prepared and stained with haematoxylin and eosin. These sections are analyzed by a pathologist. If tumor cells are found, sections up to 15 μm thickness are prepared and transferred to P.A.L.M.® membrane slides (P.A.L.M.® Microlaser Technologies AG, Bernried, Germany). The membrane slides had previously been treated in dry heat (4 h at 180° C.) to ensure an RNase-free surface. After transfer to the slides, the sections are air-dried for 1 min on ice and then fixed by incubation for 2 min in precooled (−20° C.) 75% ethanol. After incubation in the staining solution (1% cresyl violet acetate in pure ethanol) for 20 sec, the slides are briefly dipped in 75% ethanol and then immediately transferred to 100% ethanol. After incubation for 30 sec, the sections are air-dried for about 5 min at room temperature. All ethanol and staining solutions are precooled and kept on ice during the entire procedure. Slides are then further processed immediately or stored at −80° C. To produce samples of the highest uniformity, tissue areas containing only tumor or normal prostate duct cells (1000-3000 μm2) are microdissected and collected employing a UV-laser based P.A.L.M.® MicroBeam system (P.A.L.M. Microlaser Technologies AG) according to the manufacturer's protocols. For RNA analysis the cells are collected in 10 μl of RNA-lysis buffer (RLT). From all tumors with Gleason 4 or 5 fractions, pure Gleason 3 tumor cell collectives are isolated by LMPC.
Collected tissue samples are processed applying the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. From each sample, one microliter of the final eluted RNA volume (14 μl) is applied to analysis in an Agilent Bioanalyzer microcapillary electrophoresis system (RNA 6000 Pico Kit, Agilent, Waldbronn, Germany). As an initial quality check and for further comparisons with processed tissue, a complete fresh section of each biopsy is directly transferred to lysis buffer and processed as described above before starting the microdissections. From each selected sample, RNA amounts of 20 ng (estimated on the basis of the Bioanalyzer analysis) are reverse transcribed to cDNA in a volume of 20 μl, applying random primers and the 1st strand cDNA synthesis kit for RT-PCR (AMV) (Roche, Mannheim, Germany) according to the manufacturer's protocol. For quantitative PCR-analyses in a LightCycler instrument, 3 μl of the cDNA-reactions described above are used as a template in each reaction with primers specific for human PSA mRNA (prostate specific antigen, KLK3, GenBank: NM—001648, fragment size: 154 bp). For normalization, identical cDNA-amounts are amplified with primers specific for the mRNA of the low abundant reference gene, huHPRT (Hypoxanthine phosphoribosyltransferase, GenBank: M31642, fragment size: 231 bp). Primers can be designed by TIB MOLBIOL (Berlin, Germany) to be mRNA-specific by spanning some introns and/or overlapping exon junctions. Quantification of the PCR-products is performed on the basis of double strand-specific fluorescence (LightCycler Fast Start DNA MasterPLUS SYBR Green I PCR Kit; Roche) and analyzed with LightCycler's software package. Briefly, the crossing points of the related PCR reactions, determined by the ‘second derivative maximum’ method of the software, are first used to normalize the PSA expression to the HPRT housekeeping gene, then the resulting values are used for pairwise comparison between the corresponding tumor and normal samples from each patient. The microarrays may contain 37,531 PCR-amplified products of human cDNA clones (Human Unigene Set RZPD3.1, German Resource Center for Genome Research, Berlin). PCR products from cDNA clones are purified by isopropanol precipitation, washed in 70% ethanol, and dissolved in 3×SSC/1.5 M betaine. The DNA is spotted on epoxy-coated glass slides (Quantifoil, Jena, Germany) using the ChipWriter Pro (Virtek Vision, Waterloo, Ontario) spotter and SMP3 pins (Telechem, Sunnyvale, Calif., USA). After spotting, microarrays are rehydrated, and DNA is denatured with boiling water prior to washing with 0.2% SDS, water ethanol, and isopropanol. The arrays are dried with pressured air. Total RNA (200 ng) is amplified by T7 RNA polymerase based in vitro transcription using the Ambion (Austin, Tex.) MessageAmp kit. Resultant aRNA is quality-checked with the Agilent 2100 Bioanalyzer. For primer annealing, 2 μg aRNA is mixed with 1 μg random hexamer primer, incubated at 70° C. for 10 min and cooled on ice. The labelling reaction is performed in 12.5 μl containing 2.5 μl of 5×RT buffer (Invitrogen), 1.25 μl of 0.1 M DTT, 1 μl each of 5 mM dNTP mix (dGAT), 0.5 μl of 3 mM dCTP, 0.5 μl (20 U) of RNasin, 0.5 μl of 1 mM CY-3- or CY-5-labelled dCTP (Amersham Pharmacia Biotech) and 1 μl (100 U) of Superscript II reverse transcriptase (Invitrogen). The mixture is incubated for 1 h at 42° C., and the reaction is stopped by addition of 1.25 μl of 50 mM EDTA (pH 8.0). The RNA is removed by hydrolysis with 5 μl of 1 M NaOH at 65° C. for 10 min, followed by neutralization with 1 μl of 5 M acetic acid. The samples are purified using Microcon YM-30 columns (Millipore, Billerica, Mass.). The samples are dissolved in 30 μl 1×DIG-Easy hybridization buffer (Roche Diagnostics, Mannheim, Germany), containing 5×Denhardt's solution and 10 ng/μl Cot1-DNA (Invitrogen), heat denatured (65° C., 2 min) and hybridized to the DNA on microarrays in a hybridization chamber (overnight, 37° C.). The slides are washed with 1×SSC/0.1% SDS (15 min) and 0.1×SSC/0.1% SDS (10 min) and cleaned with 70 and 95% ethanol before drying with pressured air. Arrays are scanned with the GenePix 4000B microarray scanner (Axon Instruments Inc., Union City, Calif., USA), and spots are quantified using GenePix Pro 4.1 software (Imaging Research Inc., St. Catharines, Ontario, Canada). Normalization and data analysis are performed with GenePix Pro 4.1 or GeneSpring (Silicon Genetics, Sunnyvale, Calif.) software. Statistically significant differential gene expression is calculated using “Significance Analysis of Microarrays” (SAM) SAM v1.21 (11).
In a specified example, gene expression differences between tumor and normal tissues from ca 34 prostata cancer patients were analyzed In the comparative SAM analysis between tumor and normal tissue, 324 differentially expressed genes were found (q-value: <0.358). Of the these samples, 3 were Gleason stage 5, 4 were Gleason 6, 23 were Gleason 7, 1 was Gleason 8. Of the 324 genes, 72 were expressed higher in the tumer, and 252 were expressed lower in the tumor than in the normal tissue. Based on these data, 39 selected genes were subjected to low-density arrays and validated with RNA samples of 10 tumor:normal pairs, which have been present in the microarray analysis as well. The genes of the gene signature, presented in table 1, of the present invention were selected pursuant the grade of differential expression.
E. coli,
| Number | Date | Country | Kind |
|---|---|---|---|
| 06019274.7 | Sep 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/059566 | 9/12/2007 | WO | 00 | 3/5/2010 |
