KR101948385B1 - Novel Biomarker for Analysing Prognosis of Prostate Cancer and Their Uses - Google Patents

Abstract

The present invention provides a novel biomarker for prostate cancer prognosis analysis. The present invention is based on the finding that the expression of SPINK1 or SP8 shows an increasing or decreasing pattern in patients at risk of progression to hormone refractory prostate cancer. Thus, using the marker of the present invention, the prognosis of prostate cancer can be analyzed quickly and accurately. When the marker of the present invention is used, it is possible to predict the progression or progression to hormone refractory prostate cancer, and thus it is possible to more effectively cope with the treatment of the patient.

Description

Novel Biomarker for Analgesic Prognosis of Prostate Cancer and Their Uses < RTI ID = 0.0 >

The present invention relates to a novel biomarker for analyzing the prognosis of prostate cancer and its use.

Prostate cancer is the most common cancer in men in the United States and the second leading cause of cancer deaths in the United States, as the westernized living environment and age increase.

Local prostate cancer can be cured by surgery or radiation therapy, but recurrence after radical treatment or male hormone therapy in most cases of metastatic prostate cancer. In men with hormone therapy, about 70-80% of patients develop a prostate specific antigen (PSA) with a decrease, but after 2 years, they lose the cell death process and do not respond to male hormone therapy Progression to refractory prostate cancer. Hormone-refractory prostate cancer is a biochemically or clinically progressive stage of prostate cancer in which serum testosterone is reduced to a steady level. Biochemical recurrence of PSA is preceded by clinical progression of bone metastasis And it is generally reported that the diagnosis and death of hormone refractory prostate cancer takes about 2.5-3.2 years on average.

Since most deaths occur in the stage of hormone refractory prostate cancer in prostate cancer, progression to these hormone refractory prostate cancers is highly correlated with patient survival, and therefore the progression to hormone refractory prostate cancer If we can predict the time of onset and progression, we will be able to cope with the treatment more effectively.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Vainio P, et al. Prostate. 2012; 72: 789-802. Cai C, Wang H, He HH, Chen S, He L, Ma F, et al. J Clin Invest. 2013; 123: 1109-22. Grasso CS, Wu YM, Robinson DR, Cao X, Dhanasekaran SM, Khan AP, et al. Nature. 2012; 487: 239-43. Olmos D, Brewer D, Clark J, Danila DC, Parker C, Attard G, et al. Lancet Oncol. 2012; 13: 1114-24. Ross-Adams H, Lamb AD, Dunning MJ, Halim S, Lindberg J, Massie CM, et al. EBioMedicine. 2015; 2: 1133-44. Robinson MD, McCarthy DJ, Smyth GK. Bioinformatics. 2010; 26: 139-40. Suraneni MV, Moore JR, Zhang D, Badeaux M, Macaluso MD, DiGiovanni J, et al. Cell Cycle. 2014; 13: 1798-810. Sorensen KD, Abildgaard MO, Haldrup C, Ulhoi BP, Kristensen H, Strand S, et al. Br J Cancer. 2013; 108: 420-8. Larkin SE, Holmes S, Cree IA, Walker T, Basketter V, Bickers B, et al. Br J Cancer. 2012; 106: 157-65. Corcoran C, Rani S, O'Driscoll L. Prostate. 2014; 74: 1320-34. De Petrocellis L, Arroyo FJ, Orlando P, Schiano Moriello A, Vitale RM, Amodeo P, et al. J Med Chem. 2016; 59: 5661-83. Uysal-Onganer P, Kawano Y, Caro M, Walker MM, Diez S, Darrington RS, et al. Mol Cancer. 2010; 9: 55. Welsh M, Moffat L, McNeilly A, Brownstein D, Saunders PT, Sharpe RM, et al. Endocrinology. 2011; 152: 3541-51. Ohmuraya M, Hirota M, Araki M, Mizushima N, Matsui M, Mizumoto T, et al. Gastroenterology. 2005; 129: 696-705. Wang GP, Xu CS. World J Gastrointest Pathophysiol. 2010; 1: 85-90. Flavin R, Pettersson A, Hendrickson WK, Fiorentino M, Finn S, Kunz L, et al. Clin Cancer Res. 2014; 20: 4904-11.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately analyzing the prognosis of prostate cancer. As a result, there is a significant correlation between the expression level of SPINK1 (serine protease inhibitor Kazal-type 1) or SP8 (Sp8 transcription factor) expressed in prostate cancer tissues or cells and the prognosis of prostate cancer, The present invention has been accomplished by confirming that the prognosis of cancer is possible.

It is therefore an object of the present invention to provide a kit for prognosis analysis of a prostate cancer patient.

It is another object of the present invention to provide a method for providing information necessary for the prognosis analysis of a prostate cancer patient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a prognostic assay for a prostate cancer patient comprising a primer or a probe that specifically binds to a nucleotide encoding SPINE1 (serine protease inhibitor Kazal-type 1) or SP8 (Sp8 transcription factor) For example.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately analyzing the prognosis of prostate cancer. As a result, there was a significant correlation between the expression level of SPINK1 or SP8 expressed in prostate cancer tissues or cells and the prognosis of prostate cancer, and it was confirmed that the prognosis of prostate cancer can be determined through the marker.

The present inventors confirmed that using the marker of the present invention, the prognosis of prostate cancer can be quickly and accurately analyzed (predicted) from a sample (prostate cancer tissue or cell) collected from an individual.

The term " prognosis " in the context of the present invention encompasses the course of disease progression, in particular, disease progression, prediction of disease regeneration. Preferably, the prognosis in the present invention means the possibility that prostate cancer in a prostate cancer patient will progress to a hormone refractory prostate cancer (CRPC).

According to one embodiment of the invention, the prognosis analysis is a prediction of progression to hormone refractory prostate cancer (CRPC).

Prostate cancer is mainly treated with surgery or hormone therapy to block the synthesis of androgen and inhibit the androgen receptor, and hormone refractory prostate cancer means prostate cancer that no longer responds to conventional hormone therapy. The term " hormone refractory prostate cancer " may be interchanged or intermingled with " castration resistant prostate cancer ".

Since most deaths occur in the stage of hormone refractory prostate cancer in prostate cancer, progression to these hormone refractory prostate cancers is highly correlated with the survival of the patient, and the patient's progression to hormone refractory prostate cancer If we can predict the timing of progression, we can cope with the treatment more effectively.

The kit of the present invention includes a substance capable of measuring the expression level of a marker for prognostic analysis in a patient with prostate cancer. The term " marker for analyzing the prognosis of a patient with prostate cancer " refers to a biomolecule capable of analyzing the prognosis of prostate cancer and showing low expression or high expression in a patient at high risk of progression to hormone refractory prostate cancer.

For purposes of the present invention, the marker is SPINE1 (serine protease inhibitor Kazal-type 1) or SP8 (Sp8 transcription factor) expressed in prostate cancer tissue or cells.

For example, the gene sequence of SPINK1 is NCBI reference sequence: AF286028.1, the amino acid sequence is NCBI reference sequence: AAG00531.1, the sequence of SP8 is the gene sequence of SP8 Is the NCBI reference sequence: BC038669.1, the amino acid sequence is the NCBI reference sequence: AAH38669.1.

According to one embodiment of the present invention, the prognosis assay kit for a prostate cancer patient of the present invention comprises a primer or a probe specifically binding to a nucleotide encoding SPINK1 or SP8.

The primer or probe used in the kit of the present invention has a sequence complementary to the SPINK1 or SP8 nucleotide sequence. The term " complementary " as used herein means having complementarity enough to selectively hybridize to the nucleotide sequences described above under any particular hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from the term complementary, and the primers or probes of the present invention may include one or more mismatches mismatch < / RTI > sequence.

As used herein, the term " primer " refers to a primer that, under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) in a suitable buffer at a suitable temperature, - means strand oligonucleotide. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the above-mentioned nucleotide sequence, which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the above-mentioned nucleotide sequence, for example, by using a program for primer design (for example, PRIMER 3 program).

As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

The nucleotide sequence of the marker of the present invention to be referred to in the preparation of the primer or probe can be found in the NCBI reference sequence described above, and the primer or the probe can be designed with reference to this sequence.

According to one embodiment of the present invention, the prognostic assay kit of the prostate cancer patient of the present invention may be a gene amplification kit.

The term " amplification " as used herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315) (SEQ ID NO: 1), self-sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction CP-PCR), U.S. Patent No. 4,437,975) (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop-mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the kit of the present invention is carried out using a primer, the gene amplification reaction is carried out to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the degree of expression of the nucleotide sequence of the marker of the present invention, the amount of mRNA of the nucleotide sequence of the marker of the present invention is examined in a sample (for example, prostate tissue) to be analyzed and the nucleotide sequence And the like.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere , C. et al, Plant Mol Biol Rep, 9:..... 242 (1991); Ausubel, FM et al, Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al Anal. Biochem . 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and the cDNA can be easily synthesized using the oligo dT primer and the reverse transcriptase using such a sequence characteristic ( reference: PNAS USA, 85: 8998 ( 1988); Libert F, et al, Science, 244:.... 569 (1989); and Sambrook, J. et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the " Clenow " fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The cDNA of the nucleotide sequence of the thus amplified marker of the present invention is analyzed by a suitable method to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resulting band is observed and analyzed to examine the expression level of the nucleotide sequence of the marker of the present invention.

Therefore, when the kit for prognosis of the prostate cancer patient of the present invention is carried out based on the amplification reaction using cDNA, specifically, (i) the amplification reaction is carried out using the primer annealed to the nucleotide sequence of the marker of the present invention step; And (ii) analyzing the product of the amplification reaction to determine the degree of expression of the nucleotide sequence of the marker of the present invention.

The marker of the present invention is a biomolecule which is low-expressed or highly expressed in a patient who is at high risk of progressing to hormone refractory prostate cancer. Low expression and high expression of these markers can be measured at the mRNA or protein level. As used herein, the term " high expression " means that the expression level of a nucleotide sequence of interest in a sample to be investigated is higher than that of a patient having a low risk of progression to hormone refractory prostate cancer, Quot; low expression " means that the expression level of the nucleotide sequence of interest in the sample to be investigated is lower than that of the patient having a low risk of progression to hormone refractory prostate cancer. For example, expression analysis methods conventionally used in the art, such as RT-PCR or ELISA methods (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001) In the case of expression analysis, it means that the expression is analyzed to be high. For example, when analyzed according to the above-described analysis method, it was determined that the SPIK1 marker of the present invention had a high risk of progressing to hormone refractory prostate cancer in the present invention when high expression and / or low SP8 expression was detected do.

According to one embodiment of the present invention, the prognosis assay kit of the prostate cancer patient of the present invention may be a microarray.

In the microarray of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA to be applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can be varied. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The kit for prognosis of the prostate cancer patient of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Hybridization-based analysis using the probe hybridized to the nucleotide sequence of the marker of the present invention described above can predict or analyze the prognosis of prostate cancer patients.

The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin , Haptens with specific binding partners such as biotin, digoxigenin and chelating groups. Markers can be generated using a variety of methods routinely practiced in the art such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, color measurement, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biosamples. The raw sample is preferably a prostate cancer tissue or a cell. Instead of the probe, the cDNA to be analyzed may be labeled and subjected to a hybridization reaction-based analysis.

When a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. The detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate. Therefore, when the kit for prognosis of the prostate cancer patient of the present invention is carried out on the basis of hybridization, specifically (i) hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction has occurred. By analyzing the intensity of the hybridization signal by the hybridization process, the prognosis of a prostate cancer patient can be determined.

According to one embodiment of the present invention, the kit for prognosis of a patient with prostate cancer of the present invention may be an immunoassay kit comprising a binding agent that specifically binds SPINK1 or SP8 protein.

The analysis of protein expression used in the analysis of the prognosis of patients with prostate cancer in the present invention is a process for confirming the presence and expression level of the marker protein of the present invention expressed in a biological sample, ≪ / RTI > to identify the amount of protein. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip are examples of immunoprecipitation, protein immunoassay, immunohistochemical staining, The method of analysis of the invention is not limited.

In the present invention, the binding agent that specifically binds to a protein includes, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA) to be.

&Quot; Antibody " in the present invention means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Examples of the kit in the present invention include an immunochromatography strip kit, a lumenex assay kit, a protein microarray kit, an ELISA kit, or an immunological dot kit. The kind is not limited.

Through the above analysis methods, it is possible to compare the amount of antigen-antibody complex formed in the biological sample isolated from the subject patient and determine whether the expression level of the prognostic marker gene in the prostate cancer patient is significantly increased or decreased , The risk of progression to and progression to hormone refractory prostate cancer in patients with prostate cancer can be predicted.

In the present invention, the term " antigen-antibody complex " refers to a combination of a prognostic assay marker protein and a specific antibody thereof in a prostate cancer patient. The amount of the antigen-antibody complex formed is determined by the size of a signal of a detection label Lt; RTI ID = 0.0 > quantitative < / RTI >

According to another aspect of the present invention, the present invention provides a method for analyzing the prognostic value of SP8 (Sp8 Transcription Factor) expression level in prostate cancer tissue or prostate cancer cells isolated from prostate cancer patients Provides a way to provide information.

According to another aspect of the present invention, there is provided a method for detecting prostate cancer, comprising measuring the expression level of SPINK1 (serine protease inhibitor Kazal-type 1) and SP8 (Sp8 transcription factor) in prostate cancer tissue or prostate cancer cells isolated from prostate cancer patients Provides a method for providing information necessary for the prognosis of patients with prostate cancer.

Because the method and the kit described above are inventions for the same marker, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

As seen in the following examples, the level of SPINK1 expression in prostate cancer tissues was higher in patients with advanced risk of progression from advanced cancer of prostate to hormone refractory prostate cancer, Because the risk of progression from prostate cancer to hormone refractory prostate cancer is lower, this level of expression can be measured to provide information needed for the prognosis of patients with prostate cancer. For example, the level of expression of the measured marker can be compared with the value of the patient who knows the prognosis, or the expression level of the marker can be quantitatively quantified according to a certain criterion, and the prognosis of the patient can be predicted based on the numerical value.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a novel biomarker for prostate cancer prognosis analysis.

(Ii) The present invention is based on the finding that the expression of SPINK1 or SP8 shows an increasing or decreasing pattern in patients at risk of progression to hormone refractory prostate cancer. Thus, using the marker of the present invention, the prognosis of prostate cancer can be analyzed quickly and accurately.

(Iii) When the marker of the present invention is used, progression to and progression of hormone refractory prostate cancer can be predicted, so that it is possible to more effectively cope with the treatment of the patient.

Figure 1 shows the gene expression pattern of prostate cancer in the NGS cohort (n = 45).
Perform hierarchical cluster analysis to obtain gene expression patterns. Benign prostate hyperplasia (BPH), cancers of the prostate (CaP), progressive CaP, and castration-resistant prostate cancers (CRPC).
A hierarchical analysis (1,274 genes) was performed by selecting genes with at least a two-fold difference in expression relative to the average gene expression level in at least 10 tissues across all tissues. Red and green indicate high expression and low expression, respectively. NGS, Next generation sequencing.
Figure 2 shows Bland-Altman plots of genes that are differentially expressed in two prostate tissue types.
(A) BPH and local CaP, (B) local CaP and progressive CaP, and (C) a mean expression value and fold ratio between progressive CaP and CRPC.
The red dot represents a statistically significant gene in the GLM likelihood test using EdgeR software. The cut-off criterion is P-value less than 0.001. logFC, log2-transformed fold change. logCPM, log2-transformed counts per million mapped reads.
Figure 3 shows the results of analysis of genes that are differentially expressed in prostate tissue.
(a) Venn diagram of gene selected by GLM likelihood ratio test using EdgeR software in long distance organization group. The gene for the red circle (gene list A) represents a gene that is differentially expressed between benign prostate hyperplasia (BPH) and primary cancerous prostate (CaP). The orange circle gene (gene list B) represents a gene that is differentially expressed between CaP and locally advanced CaP. The gene for black circles (gene list C) represents a gene that is differentially expressed between progressive CaP and castration-resistant prostate cancer (CRPC). A cut-off criterion of a P-value less than 0.001 and a relative difference of more than 2-fold is applied to select genes with significant expression differences between the two groups.
(b) Represent the expression pattern of the selected gene in the Venn diagram. Data is represented in matrix format. Red and green indicate high expression and low expression, respectively.
Figure 4 shows the result of comparative analysis of the list of important genes in the matched sample pairs and the unpaired samples.
Generic linear model using EdgeR software Venn diagram of the gene selected through the likelihood ratio test. The blue circle genes (gene list A) represent genes that are differentially expressed between progressive CaP and CRPC in the paired samples. Genes in green circles (gene list B) represent genes that are differentially expressed between progressive CaP and CRPC in unpaired samples. Apply a cut-off criterion of P-value less than 0.001 and a relative difference of 2 or more to select genes with significant expression differences between the two groups.
Figure 5 shows the expression level of prostate cancer in the open data set (GSE28403).
The two groups of box plots compare the expression levels of genes involved in CRPC progression in the advanced CaP and CRPC groups. The access number in parentheses refers to the probe ID of the microarray platform. # Is associated with changes in mean gene expression in the NGS cohort. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. NGS, next generation sequencing. *, P < 0.05. The P-value is obtained by t-test for two samples.
Figure 6 shows the expression levels of prostate cancer in the open data set (GSE32269).
The two groups of box plots compare the expression levels of the genes involved in CRPC progression in the local CaP and CRPC groups. The access number in parentheses refers to the probe ID of the microarray platform. # Is associated with changes in mean gene expression in the NGS cohort. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. NGS, next generation sequencing. *, P < 0.05. **, P < 0.001. The P-value is obtained by t-test for two samples.
Figure 7 shows the expression levels of prostate cancer in the open data set (GSE35988).
The two groups of box plots compare the expression levels of genes associated with CRPC progression in the local CaP and metastatic CRPC groups. The access number in parentheses refers to the probe ID of the microarray platform. # Is associated with changes in mean gene expression in the NGS cohort. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. NGS, next generation sequencing. *, P < 0.05. **, P < 0.001. The P-value is obtained by t-test for two samples.
Figure 8 shows the results of seven differentially expressed genes in an independent cohort using real-time PCR.
Cohort agrees with non-cancer BPH, CaP, hormone-inhibited CaP, and CRPC tissue. Among them, AR, SPINK1, SP8, CEACAM20, and CEACAM22P showed the same expression pattern in the NGS cohort.
FIG. 9 shows the results of analyzing the five genes selected from the NGS cohort and the verification cohort-1 for 94 advanced CaP samples obtained from the independent verification cohort-2 and evaluating the prognostic value of each candidate gene associated with CRPC progression .
SPINK1 and SP8 represent significant predictions for CRPC progress time. The frequency of progression was significantly higher in the SPINK1 high expression group than in the SPINK1 low expression group and significantly higher in the SP8 low expression group than in the SP8 high expression group.
FIG. 10 shows gene set analysis results for genes associated with CRPC progression.
Enriched gene set terms were determined using Ingenuity Pathway Analysis software. The limit value for significance is P <0.05.
Figure 11 shows the gene network of genes associated with CRPC progression.
Investigate gene networks using 90 differentially expressed genes between progressive CaP and CRPC. Highly expressed and underexpressed genes in the CRPC subgroup are shown in red and green, respectively. The intensity of color indicates high or low expression level. Each line and arrow represents the functional and physical interactions of the regulatory direction reported in genes and literature. CaP, cancer of prostate. CRPC, castration-resistant prostate cancer.
Figure 12 shows the expression levels of prostate cancer in the open data set (GSE28403).
The rod plots compare the expression levels of genes associated with CRPC development in the advanced CaP and CRPC groups. "↓" indicates downward adjustment in the CRPC subgroup. CaP, cancer of prostate. ADV, advanced CaP. CRPC, castration-refractory prostate cancer.
Figure 13 shows the expression level of prostate cancer in the open data set (GSE32269).
The rod plots compare the expression levels of genes associated with CRPC development in the local CaP and CRPC groups. "↓" indicates downward adjustment in the CRPC subgroup. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. *, P < 0.05. The P-value is obtained by t-test for two samples.
Figure 14 shows the expression levels of prostate cancer in the open data set (GSE35988).
The rod plots compare the expression levels of genes associated with CRPC development between (A) GPL6480 and (B) GPL6848 platforms between the local CaP and CRPC groups. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. *, P &lt; 0.05. **, P &lt; 0.001. The P-value is obtained by t-test for two samples.
Figure 15 shows the expression levels of prostate cancer in the open data set (GSE37199).
Rod plots compare the expression levels of genes associated with CRPC development in blood samples from patients with good prognosis and CRPC patients. "↓" indicates downward adjustment in the CRPC subgroup. CRPC, castration-refractory prostate cancer. *, P &lt; 0.05. The P-value is obtained by t-test for two samples.
Figure 16 shows the expression level of prostate cancer in the open data set (GSE70768).
The rod plots compare the expression levels of genes associated with CRPC development in CaP and CRPC patients. CaP, cancer of prostate. CRPC, castration-refractory prostate cancer. *, P &lt; 0.05. The P-value is obtained by t-test for two samples.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

Patient and tissue samples

A total of 288 prostate tissue samples, consisting of three independent cohorts, were used in this study. The first NGS cohort used to generate RNA sequence data to identify transcript features was 8 benign prostate hyperplasia (BPH), 16 cancers of the prostate (CaP), 9 progressive CaP, and 12 CRP -resistant prostate cancers) (Table 1). Four of the CRPCs were in parallel with four advanced CaPs, and samples of CPRC and progressive CaP were obtained from the same patient (Table 2). Biopsy of the two samples was performed at each stage of their disease progression. In this cohort all samples were obtained from the primary location of the prostate and no metastatic samples were found at other distant site locations. All patients in this study were treated at Chungbuk National University Hospital. All tumor samples were taken under the patient's consent for tissue sample donation and testing. In this study, CRPC was defined as three consecutive increases in PSA despite low serum testosterone levels (<50 ng / mL).

Verification cohort-1 includes 58 non-cancer BPH controls, 62 locally advanced or progressive CaP cases, 14 hormone-suppressed CaPs, and 15 CRPC cases . Hormone suppression CaP implies a case of receiving androgen deprivation therapy (ADT) without any indication for CPRC. Verification cohort-2 consists of 94 patients with locally advanced or progressive disease who received post-operative ADT.

Clinical characteristics of patient and control group in NGS cohort, validated cohort-1, and cohort-2 Characteristic NGS cohort Verification Cohort -1 Verification Cohort-2 BPH control Local CaP Progressive CaP CRPC BPH control Locally advanced or progressive CaP Hormone suppression CaP CRPC Locally advanced or progressive CaP No. 8 16 9 (4: paired) 12 (4: paired) 58 62 14 15 94 age
(Age; range) 65.1
(54-70) 68.1
(54-75) 74.9
(69-82) 73.7
(59-82) 71.5
(54-90) 71.4
(48-85) 72.2
(52-78) 69.6
(53-85) 69.9
(52-86) The PSA ± standard deviation (ng / ml) 1.8 ± 0.4 15.1 ± 7.9 332.8 ± 276.0 62.3 ± 56.0 1.7 ± 1.3 387.9 ± 1239.3 9.8 ± 15.7 193.6 ± 387.3 277.8 ± 959.5 Operation (%) TURP 8 0 One 12 58 44 (71.0) 14 (100) 15 (100) 59 (62.8) Radical prostatectomy 0 16 8 0 18 (29.0) 0 (0.0) 0 (0.0) 35 (37.2) Gleason Score (%) 6 or less 2 0 0 5 (8.1) 0 (0.0) 0 (0.0) 0 (0.0) 7 11 3 2 20 (32.3) 2 (16.3) 3 (20.0) 42 (44.7) 8 0 0 2 14 (22.6) 3 (21.4) 1 (6.7) 17 (18.1) 9 3 6 7 21 (33.9) 8 (57.1) 8 (53.3) 32 (34.0) 10 0 0 One 2 (3.2) 1 (7.1) 3 (20.0) 3 (3.2) TNM step (%) T2 or T3, N0, M0 16 0 0 17 (27.4) 1 (7.1) 0 (0.0) 47 (50.0) T4 or metastasis 0 9 12 45 (72.6) 13 (92.9) 15 (100) 47 (50.0)

Abbreviation: NGS, Next generation sequencing.

Clinical course case 1 ^st surgery 2 ^nd surgery age Type of surgery PSA Gleonson Score TNM step Time to CRPC
(month) age Type of surgery PSA Gleonson Score TNM step Case 1 69 RRP 90.8 7 (3 + 4) T3N1M0 124.7 81 TUR-P 133.1 7 (4 + 3) T4N1M1 Case 2 74 TUR-P 379.0 7 (4 + 3) T3N1M1 26.2 78 TUR-P 166.0 7 (4 + 3) T3N1M1 Case 3 78 TUR-P 200.0 9 (4 + 5) T3N1M0 13.8 79 TUR-P 40.2 9 (5 + 4) T3N1M0 Case 4 69 TUR-P 89.0 9 (4 + 5) T4N1M0 9.3 70 TUR-P 117.3 9 (4 + 5) T4N1M0

RNA isolation and RNA-sequencing experiments

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, CA) according to the manufacturer's protocol. The quality and integrity of RNA was confirmed by visualization through agarose gel electrophoresis and UV irradiation after ethidium bromide staining. The sequencing library was prepared using the TruSeq RNA Sample Preparation kit v2 (Illumina, Calif.) According to the manufacturer's instructions. Briefly, mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads, and cDNA was synthesized. The adapter was ligated to the cDNA and the fragment was amplified by PCR. Sequencing was performed with a fair-end lead (2 x 100 bp) using Hiseq-2000 (Illumina).

RNA-seq data processing

Standard nucleotide sequence data for humans were obtained from the University of California Santa Cruz Genome Browser Gateway (assembly ID: hg19). The reference genome index was constructed using the Bowtie2-build component of Bowtie2 (ver. 2.0) and SAMtools (ver. 0.1.18). Tophat2 was applied to the tissue samples for mapping readings to the reference genome (ver. 2.0). Statistics for the mapping activity of Tophat2 are shown in Table 3. The data set generated by RNA-seq can be found in the National Center for Biotechnology Information (NCBI) database of GEO (Gene Expression Omnibus) through accession number GSE80609.

Patient ID Tissue type # mapped reads # unmapped reads Mapping ratios (%) H726 BPH 15,994,941 7,276,439 68.7% H731 BPH 15,378,262 6,553,472 70.1% H732 BPH 25,899,812 12,409,448 67.6% H735 BPH 21,519,964 9,689,804 69.0% H736 BPH 37,972,019 17,081,725 69.0% H745 BPH 46,916,461 23,849,229 66.3% H747 BPH 20,914,425 9,428,889 68.9% H750 BPH 17,710,692 8,733,686 67.0% RP097 Local CaP 15,209,797 10,128,655 60.0% RP106 Local CaP 17,086,017 11,582,275 59.6% RP166 Local CaP 16,981,289 10,695,617 61.4% RP194 Local CaP 20,157,993 13,394,333 60.1% RP280 Local CaP 30,181,363 20,417,371 59.6% RP286 Local CaP 16,995,206 11,226,446 60.2% RP342 Local CaP 25,276,203 11,994,971 67.8% RP395 Local CaP 23,834,391 11,826,713 66.8% RP432 Local CaP 27,978,757 13,528,447 67.4% RP436 Local CaP 31,739,247 14,924,579 68.0% RP437 Local CaP 28,482,081 13,917,341 67.2% RP461 Local CaP 39,460,373 19,368,623 67.1% RP463 Local CaP 38,719,122 18,278,160 67.9% RP482 Local CaP 28,591,951 14,087,891 67.0% RP496 Local CaP 22,279,531 15,050,451 59.7% RP499 Local CaP 15,906,502 10,827,342 59.5% RP264 Progressive CaP 11,973,886 4,123,158 74.4% RP484 Progressive CaP 10,250,695 3,678,987 73.6% RP553 Progressive CaP 10,407,388 3,816,624 73.2% RP588 Progressive CaP 9,873,038 3,529,706 73.7% RP621 Progressive CaP 15,008,355 3,199,191 82.4% RP160 Progressive CaP 18,827,736 4,581,680 80.4% RP516 Progressive CaP 17,828,552 5,174,192 77.5% RP542 Progressive CaP 18,819,675 5,557,045 77.2% RP62 Progressive CaP 13,406,642 4,763,494 73.8% RP160 + CRPC 18,743,673 4,813,405 79.6% RP516 + CRPC 37,488,274 14,279,668 72.4% RP542 + CRPC 9,889,280 3,503,332 73.8% RP62 + CRPC 21,217,451 5,696,825 78.8% RP170 CRPC 24,843,371 8,457,473 74.6% RP226 CRPC 18,114,401 4,353,769 80.6% RP256 CRPC 27,944,909 9,637,987 74.4% RP308 CRPC 31,224,250 11,693,276 72.8% RP407 CRPC 12,378,314 4,472,862 73.5% RP537 CRPC 22,387,454 5,035,682 81.6% RP627 CRPC 18,727,652 5,144,690 78.4% RP680 CRPC 26,522,537 8,900,425 74.9%

Open gene expression data set for validation

Five gene expression data sets of tumor samples from various disease stages, including progressive CaP and CRPC, were collected from NCBI GEO (GSE28403, GSE32269, GSE35988, GSE37199 and GSE70768) to confirm gene expression level changes between progressive CaP and CRPC Respectively. All progressive CaPs and two CRPCs were collected from distant metastatic sites in GSE28403, including four progressive CaPs and nine CRPC gene expression data, and the remaining CRPC samples were obtained at the primary site of the prostate (1) .

GSE32269 includes 22 primary localized CaP (hormone-dependent) and 29 CRPC transposon to bone (2). GSE35988 includes 59 localized prostate cancer and 35 metastatic CRPC gene expression data generated from two custom microarray platforms (3). The gene expression data set in GSE37199 was generated from 107 blood samples from 94 CaP patients (31 with good prognosis and 63 with progressive CRPC) and 9 from biological replicates and 4 technical replicates . Thus, GSE37199 includes 39 CaPs with good prognosis and 68 CRPCs (4). Finally, GSE70768 contains gene expression data from 13 CRPC and 113 primary CaP samples obtained by robotic radical prostatectomy (5). The GSE28403, GSE32269 and GSE37199 gene expression data sets were generated by Affymetrix Human Genome U133A and U133 Plus 2.0 Array, GSE35988 was generated by two custom Agilent platforms (Agilent Whole Human Genome Microarray G4112F and G4112A), and GSE70768 was generated by Illumina It was generated based on HumanHT-12 V4.0 expression bead chip. We have identified data sets that are fully compatible with RNA-seq data sets including CRPC from advanced CaP and primary locations of prostate tissue by exploring diverse data sets for CaP in public databases.

Real-time PCR for target gene verification

mRNA was measured by real-time PCR using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia). Real-time PCR was performed in a micro reaction tube (Corbett Research) using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, Calif., USA). Primers used to amplify mRNA for AR , SPINK1 , SERPINB11, SP8, CBNL , CEACAM20 , CEACAM22P and GAPDH from tissues were as follows.

gene The forward direction (5'-3 ') The reverse direction (5'-3 ') AR TCC ATC TTG TCG TCT TCG G TGT GAC ACT GTC AGC TTC TG SPINK1 GAA CTT AAT GGA TGC ACC AAG TCA GCA AGG CCC AGA TTT TT SERPINB11 TTG TAA AGG AGC CGC AGA TG GGT TGA AGA GAT CTG TCA CC SP8 ATG CTT GCC GCT ACC TGTA AGA CTG GAA CCC ACT ACG TT CBLN TTG TAA AGG AGC CGC AGA T CAT TAA ACT GAC CTG GAT GG CEACAM20 AG CCT CGC TTT GTA CCG TA AGA AGG GCA TCT CGA GCT TC CEACAM22P GAC CAG TAA TCT CCA GAG AT AGC AGA CCA GAG TGA GGT TG GAPDH CAT GTT CGT CAT GGG TGT GA-3 ATG GCA TGG ACT GTG GTC AT

AR , SPINK1 , SP8, CEACAM22P and GAPDH were amplified from prostate cancer tissue samples under the following conditions: denaturation for 2 seconds at 96 DEG C followed by 2 seconds at 96 DEG C, annealing at 60 DEG C for 20 seconds, and 72 DEG C Repeat 45 cycles to extend for 20 seconds. SERPINB11 , CBLN , and CEACAM20 were amplified under conditions of 1 cycle of 2 seconds at 96 deg. C, followed by denaturation at 96 deg. C for 2 seconds, annealing at 54 deg. C for 20 seconds, and extension at 72 deg. C for 20 seconds . And the temperature was increased by 1 占 폚 per 45 seconds, and the melting program was performed at 65 占 폚 and 95 占 폚. Expression of the genes was normalized to GAPDH. All samples were repeated three times.

Statistical analysis

Statistical analysis was performed using R language environment (ver. 3.0.1) and SPSS software (ver. 23).

Experiment result

Finding hormone refractory prostate cancer predictors from prostate cancer tissue

Transcriptome profiling was performed on 45 prostate samples in the NGS cohort to find the gene set associated with each progression in CaP. To assess the molecular characteristics of different sample groups, a hierarchical clustering analysis was first applied to the RNA-seq data. Unsupervised hierarchical clustering analysis of gene expression data was performed on three major sample clusters: BPH (normal), local CaP (T2 or T3 N0 M0), progressive CaP (T4 or N1 / M1) (Fig. 1). Unexpectedly, the progressive CaP and CRPC samples were clearly indistinguishable from one another, indicating that there are a small number of molecules presenting significant differences between the progressive CaP and CRPC tissue types compared to other prostate tissues.

Next, we identified genes that are differentially expressed among four types of prostate tissue types (BPH, local CaP, progressive CaP, and CRPC). The GLM likelihood ratio test identifies three genes that are differentially expressed between successive stages of CaP (ie, BPH versus local CaP, local CaP versus advanced CaP, and progressive CaP versus CPRC) P < 0.001, Fig. 2). As described earlier in the hierarchical cluster analysis, only 90 genes were found to have statistically significant differences between advanced CaP and CRPC.

Three gene lists were compared through a comparison of the Venn diagram (Fig. 3a). The gene lists &quot; A &quot;, &quot; B &quot;, and &quot; C &quot; represent differentially expressed genes between BPH and local CaP, local CaP and progressive CaP and the progressive CaP and CRPC groups, respectively. A comparison of the three gene lists found a number of different patterns: only A (2,445 genes), A∩B (95 genes), unique B (403 genes), B∩C (12 genes) , Only C (50 genes), C∩A (27 genes), and A∩B∩C (1 gene) (Figure 3b). Genes belonging to the unique A and only B categories exhibited expression patterns associated with tumor formation and advanced progression, respectively, while genes belonging to the unique C category exhibited expression patterns associated with CRPC development. The genes in the A and B categories exhibited a common expression pattern associated with tumor formation and stage progression, and the genes in the B and C categories exhibited common expression patterns related to step progression and CRPC development in CaP. AR was the only gene associated with tumor formation, stage progression and CRPC development (Figure 3). When the expression level of AR was evaluated at each stage of progression, the expression of AR was markedly decreased in tumorigenesis of local CaP, and the expression was greatly increased in progression to progressive CaP and CRPC (FIG. 2). Indicating that AR is an important mediator with diverse activities in CaP progression.

Among the samples of the NGS cohort, there were four pairs of progressive CaP and CRPC samples, each pair from the same patient. These paired samples were used to identify genes that were differentially expressed between progressive CaP and CRPC, and a comparative analysis of important gene lists was performed on paired samples and unpaired samples. Assessment of expression differences between progressive CaP and CRPC resulted in statistical significance (309 and 182 genes, respectively, in the paired and unpaired samples (P <0.001 by GLM likelihood ratio test). As a result of comparing these two types of gene lists, 15 genes were common (Fig. 4). Two of these genes ( AR and SPINK1) were overexpressed in CRPC compared to the progressive CaP group and 10 genes (ie, ALOX15B , ANPEP , CBLN2 , CEACAM20, CEACAM22P , SERPINB11 , SNCA , SP8, TRPM8 and WNT11 ) (Table 5). The remaining genes (i.e., KLK1, PGC and PPFIA2 ) showed different expression patterns in paired and unpaired samples.

A paired sample is a case in which a human sample from a prostate cancer patient is a sample of the same person, and a sample in which both a first prostate cancer human sample and a CRPC sample are present in the same person, and an unpaired sample There is CRPC sample, but there is no first prostate cancer sample of the same person. This is the first time that a tumor has recurred after the first tumor operation and the hormone therapy is finally given to the patient. Samples are very difficult to obtain, and it seems that there are few restrictions on the same person. The present inventors obtained these samples and studied them. In order to distinguish them, paired samples and unpaired samples were separated and tested.

Log2-transformed fold change values of genes that were differentially expressed between progressive prostate cancer and CPRC in paired and unpaired samples gene logFC
(CRPC vs. ADV, paired samples) logFC
(CRPC vs. ADV, unpaired sample) One ALOX15B (arachidonate 15-lipoxygenase, type B) -3.778 -7.072 2 ANPEP (alanine aminopeptidase) -2.962 -3.730 3 AR (androgen receptor) 3.847 1.826 4 CBLN2 (cerebellin 2 precursor) -3.271 -6.687 5 CEACAM20 (carcinoembryonic antigen related cell adhesion molecule 20) -4.196 -6.430 6 CEACAM22P (carcinoembryonic antigen-related cell adhesion molecule 22, pseudogene) -2.867 -6.414 7 KLK1 ( Kallikrein 1) -3.447 8.286 8 PGC (progastricsin) -3.705 4.067 9 PPFIA2 (PTPRF interacting protein alpha 2) 3.414 -3.188 10 SERPINB11 (serpin family B member 11) -3.490 -6.513 11 SNCA (synuclein alpha) -2.528 -3.057 12 SP8 (Sp8 Transcription Factor) -5.151 -5.363 13 SPINK1 (serine protease inhibitor Kazal-type 1) 4.259 4.355 14 TRPM8 (transient receptor potential cation channel subfamily M member 8) -2.852 -6.156 15 WNT11 (Wnt family member 11) -2.587 -3.605

logFC, log2-transformed fold change; CRPC, castration-refractory prostate cancer; ADV, advanced prostate cancer.

Assessment of CRPC development-related genes and their prognostic utility in an independent patient cohort

Using the 12 genes with markedly varying expression levels during CRPC progression (i. E., Two genes were overexpressed and ten genes were low expression), the genes were identified in an open gene expression data set. Comparing gene expression between progressive CaP and CRPC samples in GSE28403, most gene expression changes correlate with our cohort, with some exceptions. Some genes, including AR, show statistical significance, but this may be due to under-sampling and mixed biopsy sites in the data set (Figure 5). We also performed validation on other data sets that further included samples for local CaP and CRPC (GSE 32269). Similarly, most of the genes correlated with our NGS cohort in changes in local CaP and CRPC expression, and some genes including AR were overexpressed (Fig. 6). Gene expression analysis in GSE35988, another data set containing samples for 59 local CaPs and 35 metastatic CRPCs, showed expression changes associated with our NGS cohort in all but one gene, compared with other patient cohorts And more genes were statistically significant (FIG. 7). However, this result may not be an accurate verification because the organization acquisition locations vary in the published data set and are not exactly coincident with our cohort.

Expression levels were analyzed using gene expression data based on the results of RT-PCR experiments on validation cohort-1 to assess expression level differences for genes associated with progressive CaP to CRPC progression. Of the 10 genes, ALOX15B , ANPEP , SNCA , TRPM8 And WNT11 have been known to be related to advanced CaP (7-12). Thus, the present inventors selected two genes (AR and SPINK1) that increase expression and five genes ( SERPINB11 , SP8, CBLN2 , CEACAM20 , CEACAM22P ) whose expression is decreased. Measurement of expression levels in BPH and CaP samples revealed that SERPINB11, CEACAM20, and CEACAM22P were differentially expressed in two tissues (FIG. 8). On the other hand, the changes in gene expression during various stages of progression in CaP, hormone-inhibited CaP and CRPC were measured, and SERPINB11 and The expression of multiple genes except CBLN2 was significantly different between CaP and CRPC samples (Fig. 8), consistent with the RNA-seq data from the NGS cohort. However, when confirmed in the open data set, most candidate genes showed a correlation with our NGS cohort and expression changes, and their significance showed a clear difference in the public data set and RT-PCR validation cohort. For example, SPINK1 did not show any significance in the open data set, but showed significantly increased expression in CPRC at validated cohort-1 (FIG. 5-8). In another example, SP8 showed opposite expression patterns in the open data set and the NGS cohort (Fig. 5), which may be due to scarce samples and mixed biopsy locations in the open data set. However, the expression changes of SP8 in the validated cohort-1 showed a statistically significant correlation with the NGS cohort (Fig. 8). These results indicate that when comparing a set of genomic data to accurately reflect the characteristics of the various stages of progression in prostate cancer, it is important to have a consistent sampling method for the samples obtained from the same biopsy location as well as the importance for a sufficient number of samples Show.

In addition, we analyzed 94 progressive CaP samples from validated cohort-2 and evaluated the prognostic values of each candidate gene associated with CRPC progression. Verified cohort-2 patients were divided into two groups according to the expression level of each candidate gene and the time to CRPC was compared (time to CRPC after androgen deprivation treatment). SPINK1 and SP8 showed significant predictions for progression time to CRPC, while many genes did not predict CRPC progression. The frequency of progression was significantly higher in the group with higher SPINK1 expression levels than the group with lower SPINK1 expression levels (log-rank test, P = 0.005; Fig. 9). Similarly, the progression rate was significantly higher in groups with lower SP8 expression levels than in groups with higher SP8 expression levels (log-rank test, P = 0.002; FIG. 9). Multivariate Cox regression analysis showed that the type of surgery, lymph node or distance metastasis and SP8 expression were independent predictors of progression from advanced CaP to CRPC (Table 6).

Multivariable Cox regression model for CRPC risk in advanced prostate cancer variable Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P Surgery type (RP versus TUR-P) 8.020 (3.277-19.631) <0.001 3.708 (1.179-11.658) 0.025 PSA (<20 vs ≥ 20 ng / mL) 4.718 (1.407-15.820) 0.012 0.880 (0.218-3.563) 0.858 Lymph node or distant metastasis (no vs. no) 8.322 (2.831-24.462) <0.001 4.924 (1.299-18.664) 0.019 Gleason Score (7 vs 8-10) 3.892 (1.451-10.439) 0.007 1.817 (0.579-5.698) 0.306 SPINK1 expression (low expression versus high expression) 3.840 (1.404-10.503) 0.009 2.356 (0.779-7.129) 0.129 SP8 expression (low expression versus high expression) 0.256 (0.101-0.646) 0.004 0.308 (0.104-0.911) 0.033

CRPC, castration resistant prostate cancer; RP, radical prostatectomy; TUR-P, transurethral resection of prostate; PSA, prostate specific antigen; HR, hazard ratio; CI, confidence interval

Biological understanding of gene expression in CRPC progression

To identify the signaling pathway to progression to CRPC, the IPA tool was used to determine the functional enrichment test and gene-to-gene network analysis of the 90 differentially expressed genes between the progressive CaP and CRPC sample groups (Fig. 3). As a result of the functional enrichment assay, genes involved in cancer, cell growth and proliferation, cell cycle, and apoptosis and survival as expected were quite large. In addition, the present inventors confirmed that there are many genes involved in immunological and inflammatory diseases (FIG. 10). Interestingly, most of the enriched functions contained AR or SPINK1, and two important tumorigenic molecules were common in CRPC samples with or without pairs in our NGS cohort, suggesting that they play an important role in CRPC development .

By examining gene-to-gene networks in genes, SPINK1 is shown to be a downstream effector of AR, resulting in AR and SPINK1 (13) (Fig. 11). SPINK1 is responsible for various functions such as abnormal morphology and proliferation of cells (14, 15) and regulates many genes involved in the matrix metalloproteinases family including MMP13 (FIG. 11). SPINK1 is overexpressed in progressive CaP and CRPC (FIGS. 3 and 8) and has a prognostic value for CRPC progression (FIG. 9), but reports that SPINK1 expression may not be an independent prognostic indicator for recurrence or progression of CaP (16), which is consistent with our results (Table 6). The inventors found HNF1A as a gene presumed to activate a tumorigenic transcriptional regulator among 90 candidate genes (Fig. 11). Many downstream effectors including the UDP glucuronosyltransferase family members (i.e., UGT1A1 , UGT1A3 and UGT2B15 ), VIL1 , AKR1C1 / AKR1C2 and ANPEP have been implicated in cancer, cell proliferation, cell or tumor morphology, It is responsible for several molecular functions including disease.

To determine the activity of several candidate genes correlated with HNF1A or AR , expression levels were examined in a data set derived from five independent patient cohorts (GSE28403, GSE32269, GSE35988, GSE37199 and GSE70768). In this assay we investigated molecules overexpressed in CRPC over the advanced CaP subgroup in our NGS cohort. Assessment of selected genes in independent cohorts showed that their expression levels were higher in CRPC than in the control subgroups with local or advanced CaP except for some exceptions (except for three in the 54 comparison groups) (Figures 12-16 ). The public data set was used to include more samples matching the previous results in the patient cohort and identify more valid genes. For example, GSE35988 included the largest samples (94 total samples: 59 local CaPs and 35 metastatic CRPCs) and 6 genes excluded from the 9 comparison groups were significantly higher in the CRPC subgroup 14A). These results indicate that AR-SPINK1 signaling and the activity of the gene network mediated by HNF1A may be a key genetic co-determinant associated with progression from advanced CaP to CRPC.

conclusion

In the present invention, mRNA was extracted from tissues of prostate cancer patients and NGS was performed. A total of 15 candidate genes were selected according to the order of expression in hormone refractory prostate cancer tissues compared to expression in mRNA before progression to hormone refractory prostate cancer (Fig. 3).

The prostate cancer patient tissue was divided into four groups, and the candidate genes were selected by comparing the groups of Gaga. The candidate genes of AR and SPINK1, which showed different expression in all groups, were found. SERPINB11, SP8, CBLN2, CEACAM20 and CEACAM22P genes, which are highly related to prostate cancer, were selected as final candidates (Fig. 8).

The gene expression of the candidate gene finally selected as a predictor of hormone refractory prostate cancer was measured using a clinical sample, and statistical analysis was conducted based on clinical information and experimental results of each sample (FIG. 9) As a result of analyzing the genes involved in the progression of hormone refractory prostate cancer, about 90 genes were associated with SPINK1 and SP8 genes (Fig. 11).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

(a) a primer or probe that specifically binds to a nucleotide that encodes SP8 (Sp8 Transcription Factor); or (b) a binding agent that specifically binds to SP8 protein.
The kit for prognosis of a prostate cancer patient according to claim 1, wherein the prognosis analysis is a prediction of progression to hormone refractory prostate cancer (CRPC).
The kit for prognosis of a prostate cancer patient according to claim 1, wherein the kit is a gene amplification kit.
The kit for prognosis of a prostate cancer patient according to claim 1, wherein the kit is a microarray.
The method of claim 1, wherein the binding agent is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer. A kit for analyzing a patient's prognosis.
The kit for prognosis of a patient with prostate cancer according to claim 1, wherein the kit is an immunoassay kit.
Comprising measuring the level of SP8 (Sp8 Transcription Factor) expression in a prostate cancer tissue or prostate cancer cell isolated from a prostate cancer patient.
8. The method of claim 7, wherein said SP8 is low expressed in a patient at risk of progression to hormone refractory prostate cancer (CRPC).
The need for analysis of the prognosis of patients with prostate cancer, including the step of measuring the expression level of SPINK1 (serine protease inhibitor Kazal-type 1) and SP8 (Sp8 transcription factor) in prostate cancer tissues or prostate cancer cells isolated from prostate cancer patients / RTI &gt;
10. The method of claim 9, wherein said SPINK1 is highly expressed in patients at risk of progression to hormone refractory prostate cancer (CRPC), and SP8 is in a patient at risk of progression to hormone refractory prostate cancer Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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