KR101774747B1 - Diagnostic methods for prognosis of non-small-cell lung cancer using pcaf snp - Google Patents

Abstract

The present invention relates to a lung cancer prognostic marker using single nucleotide polymorphism (SNP) in a PCAF (P300 / CBP-associated factor) gene and a method for predicting the survival prognosis of lung cancer using the same. More particularly, the present invention relates to a composition for predicting survival prognosis of lung cancer based on a specific SNP present in the promoter of PCAF gene, exon and the like, a kit for predicting survival prognosis of lung cancer using the same, a method for predicting microarray and survival prognosis of lung cancer.

Description

[0002] DIAGNOSTIC METHODS FOR PROGNOSIS OF NON-SMALL-CELL LUNG CANCER USING PCAF SNP [0003]

The present invention relates to a single nucleotide polymorphism of PCAF (P300 / CBP-associated factor) for prediction of prognosis of non-small cell lung cancer (NSCLC), and more specifically, to SNP of rs17006625 The present invention relates to a method for diagnosing and predicting the prognosis affecting the survival outcome of non-small cell lung cancer and its use.

Globally, lung cancer accounts for 1,600,000 new cases each year, of which 1.4 million die. This accounted for 13% of all cancer cases and accounted for 18% of cancer-related deaths. In 2010, lung cancer deaths increased to 1,500,000, accounting for 19% of all cancer-related deaths.

Histologically, about 80% are non-small-cell lung cancer, 32-40% of whom are adenocarcinoma including bronchoalveolar carcinoma (BAC), 25-30% Squamous cell carcinoma, and 8-16% of large cell neuroendocrine carcinoma. More than 65% of all non-small cell lung cancers are found to be completely inoperable from the time of diagnosis

Diagnosis of NSCLC is performed by examination of pathologists of suspected tissues such as biopsy samples. After NSCLC diagnosis, the patient's illness is assigned to the patient's overall health status and severity of symptoms such as age, coughing and dyspnea, prognosis (recovery opportunity) using specific types of NSCLC and cancer staging. The progression stage considers the size of the tumor and whether the tumor is present only in the lung or diffuse elsewhere in the body. Specific treatment options for NSCLC patients are selected based on the above considerations, and the cancer progression stage is an important factor for treatment selection. Patients with early NSCLC can potentially be cured by surgical resection to remove the tumor, but current diagnostic aspects can not predict which patient will recur after surgery.

Also, because cancer often recurs locally or transitions from native cancer tissue to distant tissues and organs, which of the patients with early cancer need to undergo drug treatment after surgical removal of their primary tumor It is important to reveal. This is particularly important in patients with early NSCLC.

The ideal treatment for lung cancer is early detection and removal of the cancer by surgery. However, early diagnosis is difficult because early diagnosis of lung cancer has progressed to such an extent that more than half of the patients can not undergo surgery. In addition, after the onset of lung cancer, especially in patients with non-small cell carcinoma, if surgery has not progressed enough, surgery will be performed first, but only 30% can perform radical resection. The 5-year survival rate, which depends on the degree of cancer progression, was recovered in all patients who underwent curative resection, including squamous cell carcinoma (37%), adenocarcinoma (27%) and large cell carcinoma (27%). The majority of these patients relapse after surgery and become more aggressive and die. Early NSCLC prognosis can not be confirmed with sufficient accuracy to direct more aggressive therapies for patients with a high likelihood of recurrence as a current clinical diagnostic method.

Therefore, it may be necessary to identify patients with high risk of early NSCLC who have to undergo chemotherapy or have generally reevaluated treatment findings, and can determine the prognosis of NSCLC so that it is easy to decide whether to use additional treatment options The survival rate can be remarkably increased.

The present inventors selected 1,969 potential functional polymorphisms from 1,151 genes involved in the development and progression of NSCLC cancer and analyzed the genotypic characteristics of 166 non-small cell lung cancer patients who underwent curative resection using a gene chip (Affimetrix Inc., USA) To complete genetic polymorphisms associated with the prognosis of patients with early stage non-small cell lung cancer (NSCLC) through log-rank test in overall survival (OS) and disease free survival (DFS) .

Korean Patent Publication No. 10-2011-0009609 Korean Patent No. 10-0894322 Korean Patent No. 10-1307660 Korean Patent No. 10-1219794

It is an object of the present invention to provide a composition for markers for diagnosis and prediction of lung cancer prognosis, which contains a single base polymorphism (SNP) of a gene of PCAF (P300 / CBP-associated factor).

Another object of the present invention is to provide a kit for predicting the survival prognosis of lung cancer patients comprising the composition of the present invention.

Another object of the present invention is to provide a microarray for predicting the survival prognosis of lung cancer patients comprising the composition of the present invention.

Another object of the present invention is to provide a method for providing information for predicting the survival prognosis of lung cancer patients.

In order to solve the above problems, the present invention provides a composition for markers for diagnosing and predicting lung cancer prognosis, which contains a single base polymorphism (SNP) of a gene of PCAF (P300 / CBP-associated factor).

Preferably, the single nucleotide polymorphism of the PCAF gene is rs17006625A> G polymorphism in the nucleotide sequence shown in SEQ ID NO: 1. The polynucleotide is preferably, but not limited to, at least 20 contiguous bases, more preferably 20 to 100 contiguous bases.

The lung cancer may be squamous cell carcinoma, small cell carcinoma, adenocarcinoma, large cell carcinoma or non-small cell carcinoma, preferably non-small cell carcinoma. Most preferably, the patient is treated with surgery-resected lung cancer.

The present invention can provide a kit for predicting the survival prognosis of a lung cancer patient or a microarray for predicting survival prognosis of a lung cancer patient comprising the above-described composition as another embodiment.

The present invention provides a method for providing information for predicting the survival prognosis of a lung cancer patient, comprising the step of confirming the rs17006625A> G polymorphism of the gene of PCAF, from a nucleic acid extracted from a lung cancer patient.

At this time, when the genotype of the rs17006625 polymorphism of the PCAF gene is GG, it is determined that the survival prognosis is low. Most preferably, the present invention relates to providing information on the prognosis of non-small cell carcinoma.

As described above, the present invention provides a variety of uses based on the novel function of the rs17006625A> G polymorphism of the gene of PCAF as lung cancer, most preferably as a non-small-cell lung cancer prognostic marker do.

The prediction technique of lung cancer survival prognosis according to the present invention can increase the survival rate of lung cancer patients by easily evaluating the prognosis of patients with lung cancer and targeting the means and treatment for selecting and evaluating treatment methods.

Figure 1 is a Kaplan-Meier plot of overall survival (OS) and disease free survival (DFS) according to genotype (P: multivariate Cox ratio risk model, recessive model)

The terms used in the present invention are defined as follows.

Prognosis refers to the progression and cure of disease, such as lung cancer-induced death or the likelihood of progression, including onset, recurrence, metastatic spread, and drug resistance, for example neoplastic diseases such as lung cancer. For the purpose of the present invention, the prognosis refers to the risk of developing lung cancer, preferably non-small cell lung cancer, and the survival prognosis after onset, preferably a patient who has undergone surgical resection of lung cancer, more preferably, Of patients.

"Prediction" refers to the determination of the likelihood of a patient developing lung cancer, and preferably or non-favorably responding to therapies such as chemotherapy or radiotherapy to treat a patient, for example, a particular therapeutic agent, and / Elimination by surgery, and / or the likelihood and / or likelihood of survival after chemotherapy for a certain period of time without cancer recurrence. The predictive method of the present invention is a patient having a high risk of developing lung cancer for any particular patient, and can prevent or delay the onset of the disease through special and appropriate management, or select the most appropriate treatment method for a patient with lung cancer, Lt; / RTI > The predictive method of the present invention can be used to identify whether a patient is responding favorably to treatment regimens, such as, for example, a prescribed treatment or combination, surgical intervention, chemotherapy or other prescribed treatment regimen, It is possible to predict whether or not survival is possible.

"Genetic polymorphism" refers to a case where a genetic variation occurs in at least 1% of the population. The insertion, deletion, or substitution of a single nucleotide in DNA is called single nucleotide polymorphism (SNP).

The term "polymorphism " refers to a sequence in a sequence of genes that varies within a cluster. Polymorphisms consist of different "alleles ". The arrangement of this polymorphism can be confirmed by its position in the gene and the different amino acids or bases found therein. These amino acid variations are the result of two possible mutant bases, C and T, which are two different alleles. Since the genotype is composed of two different distinct alleles, any of the various possible variants can be observed in any individual (e. G., CC, CT or TT in this example). The individual polymorphisms are also known to those skilled in the art and are used in, for example, the Single Nucleotide Polymorphism Database (dbSNP) of the Nucleotide Sequence Variation of the nucleotide base mutations available on the NCBI website. ("Reference SNP", "refSNP", or "rs #").

"Single nucleotide polymorphism (SNP) refers to the diversity of DNA sequences that occur when a single nucleotide (A, T, C or G) in the genome is different between members of a species or between individual chromosomes For example, differences in single bases such as three DNA fragments of different individuals (eg, AAGT [A / A] AG, AAGT [A / G] AG, and AAGT [G / Within a population, SNPs are defined as minor allele frequencies (MAFs), which are the frequencies of allele frequencies (MAFs). (Deletion) or addition (insertion) of a polynucleotide sequence in the polynucleotide sequence of the polynucleotide sequence of the polynucleotide sequence of SEQ ID NO: It is possible to cause a change in the translation frame.

The term "genotype" refers to a specific allele of a particular gene in a cell or tissue sample.

An "allele" or "allele" is one of two or more alternative forms of a gene that occupy the same chromosomal locus.

"Allele frequency" refers to the frequency (percentage or percentage) that an allele is present within an individual, within a lineage, or within a group of lines. The allele frequency in the system or population can be estimated by averaging the allele frequency of a sample of individuals from the system or population.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. Among them, the present invention is particularly useful for diagnosis of solid pancreaticobiliary tumors of the pancreas. The present invention includes predicting the progression and metastasis of lung cancer, preferably non-small cell lung cancer recurrence. Therefore, it is very important to accurately predict the recurrence and progression of non-small-cell lung cancer, and factors that can predict the response of treatment while supplementing clinical indicators such as tissue differentiation and stage are needed. The PCAF (P300 / CBP-associated factor (SNP) can be used as a diagnostic tool for non-small cell lung cancer. That is, the polymorphism measurement of these genes can be used as an indicator (diagnostic marker) for predicting the differentiation, stage, and progression of non-small cell lung cancer.

"Diagnostic markers or diagnosis markers are substances that can distinguish cells with non-small cell lung cancer from normal cells. It is a polypeptide or nucleic acid that shows an increase pattern in non-small cell lung cancer cells compared to normal cells MRNA, etc.), lipids, glycolipids, glycoproteins, sugar (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

The term " marker for predicting the survival prognosis of a patient with lung cancer "means a marker having a polymorphism capable of predicting the risk of lung cancer, the cure of the onset of lung cancer or the progress of lung cancer, preferably the nucleotide described above. In addition, the patient refers to a patient for discriminating the risk of lung cancer or a patient who has undergone surgical resection of lung cancer, particularly lung cancer. The patient who has undergone surgical resection of the lung cancer preferably refers to a patient who has undergone surgical resection of lung cancer such as non-small cell carcinoma, squamous cell carcinoma, adenocarcinoma or large cell carcinoma. More preferably, do.

"Danger" refers to a statistically high incidence of a disease or condition in a subject having a particular polymorphic allele, compared to the incidence of a disease or condition in a member of the individual that does not possess the particular polymorphic allele.

The term "functional equivalents" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, a net effect that does not result in various functional dissimilarities between the reference and subject sequences, ≪ / RTI > and the nucleotide sequence of the mutated mutant sequence. Preferably, the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, most preferably about 95% identity. For the purposes of the present invention, sequences with substantially equivalent biological activities and substantially equivalent synthetic features are treated as substantial equivalents.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The disease to be diagnosed in the present invention is a disease associated with lung cancer.

The lung cancer may preferably be non-small cell lung cancer (NSCLC), and the non-small cell lung cancer includes squamous cell cancer, adenocarcinoma, large cell carcinoma, or squamous cell carcinoma. More preferably squamous cell cancer.

Patient refers to a patient with lung cancer, ie squamous cell, small cell, adenocarcinoma, large cell carcinoma or non-cell carcinoma. Preferably, the subject includes patients who have undergone surgical resection of lung cancer, i.e. squamous cell, small cell, adenocarcinoma, large cell carcinoma or non-cell carcinoma.

Non-small cell lung cancer accounts for more than 75% of all lung cancer, with an average 5-year survival rate of 15%. The high mortality rate of non-small cell lung cancer is associated with a high proportion of patients with some unresectable tumors (Parkin DM, et al., Cancer J Clin 55: 74-108, 2005). It has been reported that many of the surgically resected patients die from recurrence of cancer even though they have a good prognosis in patients with non-small cell lung cancer of the resectable stage (Arriagada R, et al., N Engl J Med 350: 351-60, 2004). Therefore, when prognostic factors are used for patients with non - small cell lung cancer, it is possible to target the evaluation and prognosis of the prognosis and the selection and treatment of the treatment method, and thus it is expected that the personalized treatment of patients with non - small cell lung cancer will be possible. The aim of this study was to evaluate the prognosis of patients with non-small cell lung cancer.

The present invention relates to the use of a PCAF (P300 / CBP-associated factor) gene polymorphism as a diagnostic and prediction marker for non-small cell lung cancer (NSCLC) Prognosis was related to prognosis. The prognosis includes progression, recurrence and metastasis of non-small cell lung cancer.

<PCAF prognostic markers>

Therefore, the present invention relates, in one aspect, to the use of the PCAF gene as a diagnostic marker for lung cancer, preferably non-small cell lung cancer prognosis.

More specifically, the present invention relates to the use of p300 / CBP-related PCAF gene polymorphism (SNP) rs17006625 A> G for the diagnosis and prediction of non-small cell lung cancer prognosis and its use.

The PCAF protein can be obtained from known, for example, known human-derived sequences from known DBs, including, but not limited to, functional equivalents thereof.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The non-small-cell lung cancer prognostic markers of the present invention show the same results in repeated experiments with genes whose expression changes either directly or indirectly with the onset of lung cancer, preferably non-small cell lung cancer, Are highly reliable markers that are very large when compared to a few, and are less likely to give false results. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The present inventors investigated the polymorphism of the PCAF gene in 166 patients who underwent curative resection of non-small cell lung cancer to determine the relationship between genotype and overall survival (OS) and disease-free survival (DFS) Respectively.

The term "total survival (OS)" refers to the period from the day of surgery to the day of death due to any cause or the last follow-up date. DFS refers to the day from surgery to the day of recurrence or death for any cause. Analysis showed that rs17006625A> G polymorphism was significantly associated with overall survival and disease-free survival in patients with non-small cell carcinoma. rs17006625 GG genotype showed OS and DFS worse than rs17006625 AA or AG genotype.

Therefore, the PCAF rs17006625A> G SNP of the present invention can be regarded as an independent prognostic marker for patients with non-small cell lung cancer undergoing surgical resection. An analysis of this may help to determine the group of patients at high risk for poor disease non-small-cell lung cancer prognosis and may help in the therapeutic determination of non-small-cell lung cancer.

As one embodiment of the present invention, the present invention relates to a marker for survival prognosis (diagnosis) of a lung cancer, preferably a non-small cell lung cancer patient comprising a polymorphic site of a PCAF gene and a composition for a marker comprising the same.

The present invention provides a polynucleotide comprising 20 to 100 consecutive DNA sequences comprising the base, wherein the 464th base is A or C in the polynucleotide of SEQ ID NO: 1; Or a complementary polynucleotide thereof, to a marker for predicting the survival prognosis of a lung cancer patient and a composition for a marker comprising the same.

The polynucleotide or its complementary polynucleotide according to the present invention may be composed of at least 20, preferably 20 to 100, more preferably 20 to 50 contiguous bases.

The polynucleotide or its complementary polynucleotide is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a single base polymorphism in the nucleotide sequence. A polymorphic site is a site in a polymorphic sequence where a single base polymorphism occurs.

Single nucleotide polymorphisms can be included in coding sequences of genes, non-coding regions of genes, or in intergenic regions between genes. SNPs in the coding sequence of a gene do not necessarily cause changes in the amino acid sequence of the target protein due to the degeneracy of the genetic code. SNPs that form the same polypeptide sequence are called synonymous (also called silent mutations) and SNPs that form other polypeptide sequences are said to be non-synonymous. Non-consensual SNPs can be missense or nonsense, and mismatch changes produce other amino acids while nonsense changes form non-mature termination codons. SNPs that are not in the protein-coding region can induce gene silencing, transcription factor binding, or non-coding RNA sequences.

In the present invention, as an example, about 49% in the promoter region of the PCAF gene, about 23% in the exons (non-similar SNPs), about 15% in the exon-intron boundary, about 7% in the 5-UTRs, SNPs are located at about 6%.

The present invention extends to a genetic approach to PCAF PCAF rs17006625 A > G.

<How to Perform>

The genotyping of the SNP of the present invention can be confirmed by sequencing analysis, sequencing analysis using an automatic sequencer, pyrosequencing, hybridization with a microarray, PCR-RELP (restriction fragment length polymorphism), PCR-SSCP single strand conformation polymorphism (PCR), SSO (specific sequence oligonucleotide), ASO (allele specific oligonucleotide) hybridization method using PCR-SSO method and dot hybridization method, TaqMan-PCR method, MALDI-TOF / MS method, RCA method rolling circle amplification, HRM (high resolution melting), primer extension, Southern blot hybridization, dot hybridization, and the like.

Further, the results of the SNP polymorphism can be statistically processed using statistical analysis methods commonly used in the art, such as Student's t-test, Chi- continuous variables, categorical variables, odds ratios, and 95% confidence intervals, obtained through linear regression analysis, linear regression line analysis, and multiple logistic regression analysis, % Confidence interval, and so on.

The agent capable of detecting the SNP contained in the marker for lung cancer, preferably non-small cell lung cancer survival prognosis prediction (diagnosis) of the present invention and the marker composition containing the same, comprises the polynucleotide of SEQ ID NO: 1, a polynucleotide consisting of 20 to 100 contiguous bases comprising rs17006625 A > G SNP, a complementary polynucleotide thereof, a primer specifically hybridizing with the polynucleotide, and a probe.

In the present invention, a primer or a probe that specifically hybridizes with the polynucleotide or its complementary polynucleotide is allele-specific.

Allele-specific refers to hybridizing specifically to each allele, i.e., hybridizing such that the base of the polymorphic site present in the polymorphic sequence can be specifically discriminated. Here, hybridization is usually carried out under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher.

In the present invention, a probe means a hybridization probe, and means an oligonucleotide capable of binding sequence-specifically to a complementary strand of a nucleic acid. The hybridization conditions show a significant difference in the intensity of hybridization between alleles and should be sufficiently stringent to hybridize to only one of the alleles. Preferably, the probe of the present invention aligns with the polymorphic site of the polymorphic sequence. This can lead to good hybridization differences between different allelic forms. The probe can be used in a kit such as a microarray for predicting the survival prognosis of lung cancer by detecting alleles, a prediction method and the like. Important probes can be labeled for detection and can be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.

In the present invention, the primer can form a base pair with a template complementary to a base sequence having a short free 3 'hydroxyl group, and functions as a starting point for template strand copying Quot; short sequence &quot; The appropriate length of the primer may vary depending on the purpose of use, but is generally comprised of 15 to 30 bases. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primers can be hybridized to a DNA sequence containing a polymorphic site to amplify a DNA fragment containing the polymorphic site. The primer of the present invention can be used in a kit such as a microarray for predicting the survival prognosis of lung cancer and a prediction method by detecting alleles.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The present invention relates, in another embodiment, to a microarray for predicting the survival prognosis of lung cancer, preferably non-small cell lung cancer patients, comprising said composition.

The microarray may be composed of a conventional microarray except that it comprises the polynucleotide, primer or probe of the present invention.

The hybridization of nucleic acids on a microarray and the detection of hybridization results are well known in the art. The detection may be performed, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal including a fluorescent substance, such as Cy3 and Cy5, and then hybridizing on the microarray, The hybridization result can be detected.

The microarray method can simultaneously study the expression of RNA in thousands or even tens of thousands of genes in tumors, enabling more comprehensive insights into the molecular basis of human disease. It can also be used to assess gene expression patterns, clinical outcomes, and response to chemotherapy regimens in tumor classifications.

In another aspect, the present invention relates to a kit for predicting the survival prognosis of a lung cancer patient comprising the composition.

The kit of the present invention can be used to predict the prognosis of lung cancer by identifying the PCAF rs17006625A> G polymorphism site (SNP), a predictive marker for the survival prognosis of lung cancer. Kits for predicting the survival prognosis of lung cancer of the present invention may include one or more other component compositions, solutions or devices suitable for the assay as well as polynucleotides, primers or probes for identifying SNPs of PCAF rs17006625A> G .

In one embodiment, the kit of the present invention may be a kit containing the necessary elements necessary for performing PCR. The PCR kit may contain test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerases and reverse transcriptase enzymes, as well as specific polynucleotides, primers or probes specific for the SNPs. , DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

<Diagnosis and Information Delivery Method>

On the other hand, the present invention, on the other hand, provides a method for diagnosing lung cancer, preferably non-small-cell lung cancer prognosis, comprising measuring rs17006625A> G SNP expression level and / or variation, preferably frame shift variation, Diagnosis and prediction method, or a method for providing information therefor.

The method also provides useful information for the development of a biodegradable polymer patch having an anti-cancer agent for preventing recurrence or metastasis after lung cancer surgery.

In one embodiment, the method of the present invention comprises

Measuring the rs17006625A> G SNP expression level of the PCAF gene from the biological sample isolated from the patient; And comparing the rs17006625A> G SNP expression level or the level of the protein encoded by the gene with the expression of the gene of interest in a normal control sample, to provide information for diagnosing and predicting the prognosis of lung cancer, preferably non-small cell lung cancer &Lt; / RTI &gt;

In this case, the PCAF rs17006625 A> G polymorphism in the biological sample isolated from the patient is characterized in that the risk ratio of the overall survival and disease-free survival of the patient group having the GG genotype is much higher than that of the AA or AG genotype. In other words, if the genotype of the SNP is GG, it can be diagnosed and predicted that the survival prognosis is bad.

The diagnostic methods relate to examining the expression characteristics of particular markers, particularly in relation to non-small cell lung cancer, and the methods disclosed herein are useful for obtaining data and information useful in assessing appropriate or effective therapies for the treatment of patients with non- Efficient, and cost-effective means. &Lt; RTI ID = 0.0 &gt;

The nucleic acid of a lung cancer patient can be obtained from a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine obtained from these lung cancer patients, and the nucleic acid sample is synthesized from DNA, mRNA or mRNA lt; / RTI &gt;

The nucleic acid of the lung cancer patient can be carried out by conventional methods such as phenol / chloroform extraction method and protease K treatment method, and the target nucleic acid can be obtained by amplifying and purifying the target nucleic acid by PCR.

The present invention can also provide information for determining the dose of the anticancer agent and the method of administration necessary for preventing recurrence or metastasis after lung cancer, preferably non-small cell lung cancer surgery.

In other words, the present invention confirms PCAF rs17006625 A> G polymorphism of the PCAF gene from biological samples isolated from patients with non-small cell lung cancer and reports the type, amount and concentration of anticancer drugs to be administered after lung cancer surgery in cases of poor prognosis The method of administering the anticancer drug may be applied differently according to the type of the lung cancer.

Thus, the present invention includes all uses as PCAF PCAF rs17006625 A> G expression profile in lung cancer, preferably non-small cell lung cancer, as a lung cancer prognosis diagnostic and prediction marker.

Therefore, the prediction technique of lung cancer survival prognosis according to the present invention can increase the survival rate of lung cancer patients by easily evaluating the prognosis of patients with lung cancer and targeting the means and treatment for selecting and evaluating treatment methods.

<Examples>

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Unless otherwise indicated, nucleic acids are recorded in a 5 'to 3' orientation from left to right. The numerical ranges recited in the specification include numerals defining the ranges and include each integer or any non-integral fraction within a defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

Materials and methods

1. Experimental group

The discovery set was used for pathological phase I, II, or IIIA (micro-invasive N2) NSCLC, which received a healing surgical resection at Kyungpook National University Hospital, KNUH, between September 1998 and December 2006 Of the patients.

Genomic DNA samples from tumors and corresponding non-malignant lung tissue samples were provided by National Biobank in KNUH (Approval No. KNUHBIO_10-1016) and were supported by the Ministry of Health and Welfare. All patients received consent before surgery. All substances derived from National Biobank were obtained under the Institutional Review Board protocol.

For the independent verification set, a total of 626 patients were collected: 164 cases from KNUH, 293 cases from Seoul National University Hospital, and 169 cases from Seoul National University Bundang Hospital. Each participant received consent from all patients prior to surgery and received a study protocol approved by the review committee. All patients included in this study were Korean native Koreans. None of the patients included in the discovery and validation set received preoperative chemotherapy or radiotherapy. The pathological stage of the tumor was determined according to the international system for stage lung cancer.

2. Selection and genotyping of SNPs

SNPs were selected using a public database. Briefly, 1,784 candidate genes involved in cancer-related pathways were selected from the database of SABioscience (http://sabioscinece.com). To select all potentially functional SNPs, a public database (http://www.ncbi.nlm.nih.gov/SNP) was used. In the HapMap JPT data, a total of 4,215 SNPs with a small allele frequency ≥ 5% were captured. Of these, other SNPs can not be applied to platforms, so 969 SNPs among 1,151 genes were genotyped using Affymetrix custom-made GeneChips.

For validation, PL-1 was used as a dominant, recessive and / or co-dominant model in the discovery set using SEQUENOMS MassARRAY iPLEX assay (SEQUENOM Inc., San Diego, Calif.) And restriction fragment length polymorphism assay. 56 SNPs associated with both total survival (OS) and disease free survival (DFS) with R <0.05 were selected and genotyped.

3. Statistical analysis

Differences in genotypic distribution according to clinicopathologic factors of patients were compared using? 2 test. The OS was measured from the day of surgery to the last follow-up or dead day. Survival estimates were calculated using the Kaplan-Meier method.

Differences between OS and DFS across different genotype types were compared using log-rank tests. HR (hazard ratio) and 95% confidence interval (CI) were assessed using a multivariate Cox proportional hazards model. Age (≤ vs.> median age), gender (female vs. male), smoking status (non- vs. smokers), pathological stages (I vs. II-IIIA), and adjuvant therapy (yes vs. no). To minimize Type II errors, SNPs for validation were selected using a cut-off alpha error level of 0.10 in the discovery set. A homogeneity test was performed to compare the differences between the genotypically related HRs of the other subgroups. All analyzes were performed using statistical methods for Windows, version 9.2 (SAS Institute, Cary, NC, USA).

Example  1: Patient characteristics and clinical Predictive factor

Clinical and pathological characteristics of patients in discovery and validation sets; And OS and DFS are shown in Table 1. &lt; tb &gt; &lt; TABLE &gt; By univariate analysis, pathological stages in both sets were significantly associated with OS and DFS (both log-rank P [PL-R] ≤ 0.0001).

[Table 1]

Abbreviation: OSR, overall survival rate

DFSR, disease free survival rate

Thus, in the assertion set, age was associated with OS and DFS (PL-R for OS = 0.003 and PL-R for DFS = 0.03) for OS = 0.02 and 0.01)

Example  2 : SNPs  And survival outcome

Ii) genotypic validity of 166 with 90%, iii) 211 with minor allele frequency <5%, or iv) Hardy-Weinberg We analyzed 1,385 SNPs from 910 genes, except for 126 (P <0.05) deviations from equilibrium.

As a result, about 49% of the SNPs are located in the promoter region, 23% in the exons (nonsynonymous SNPs), 15% in the exon-intron boundaries, 7% in the 5-UTRs and 6% in the 3-UTRs Respectively.

Of the 1,385 SNPs analyzed in the discovery set, 56 SNPs were associated with both OS and DFS (PL-R <0.05) and screened for validation. When adjusted for age, sex, smoking status, pathological steps and adjuvant chemotherapy, two SNPs (p300 / CBP-related factor [PCAF] rs17006625A> G, and pericentriolar substance 1 [PCM1 ] rs17691523C> G) was found and was found to be significantly related to survival outcomes in the same direction as the discovery set in the independent assays set (Table 2 and Figure 1).

The results for PCAF rs17006625A > G are listed in Table 2 below.

[Table 2]

In particular, in combination analysis, the rs17006625 GG genotype has been shown to be associated with rs17006625 AA or AG genotype (adjusted HR [aHR] for OS = 3.24, 95% CI = 1.64-6.39, P = 7x10-4; and aHR for DFS = 2.67, 95% CI = 1.52-4.69, P = 7 × 10-4).

From these results, it can be seen that the present invention affects the survival outcome of patients who have surgically resected early stage NSCLCs (PCAF rs17006625 S).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> DIAGNOSTIC METHODS FOR PROGNOSIS OF NON-SMALL-CELL LUNG CANCER          USING PCAF SNP <130> PN1412-380 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 960 <212> DNA &Lt; 213 > PCAF DNA sequence <400> 1 tcaaaaaaat acttttgtgg ggagatattt tgagactgta taaatatctt gtcttcatca 60 aaattatact cacaggtttt agcctacaca catgaacttc cccaaatcag ttatcatatg 120 gtggctgcca aatagtgatt tttttttttt ttttttacta actaccataa ctccgtcctt 180 atttctttgt tggtgttctt gagcttttct catctccact aatttattta tgtattcatt 240 tattatttta tcaatataga ctcagatatc cttattctat ttaatgagtt atagtccctg 300 ctgtctttac tattttgatg ccttgtggct tatgttttaa agatgactta ttttgtggca 360 ttgggcagga gtggggtggt cccatgtgca cttcgtttct tgttacctaa gaaaggagtc 420 agtcatctaa caccttcttc tccttttgcc ggggcagtta tcaatccacc tcctgtggct 480 gggacaattt catacaattc aacctcatct tcccttgagc agccaaacgc agggagcagc 540 agtcctgcct gcaaagcctc ttctggactt gaggcaaacc caggtaagct cttaagaggg 600 gataagagag ggctgtgact tgctccagga gctccattac ctgaggagtg caaaaggtag 660 acttgaacca agtacagatt gtgcatgtcc aggattttaa cagctagaaa taactttgta 720 ttaggctttt ggctgtactg gttagttatt gctatcttaa aactgtaata acttattgtc 780 atagctcact catttggtgt gttgcctgag agttggctga tttcagctag tctcactcaa 840 tcttggctgg gcttggttgc aagttaccca ttgattataa tctctgtttt gggcttggct 900 tggctagagc acttaagctg aagcaactct gcttcacata tttctcataa ttttcctggg 960                                                                          960

Claims (10)

A composition for markers for the diagnosis and prediction of prognosis of non-small cell lung cancer, which comprises a single nucleotide polymorphism (SNP) of a gene of PCAF (P300 / CBP-associated factor). The method according to claim 1,
Wherein the single nucleotide polymorphism of the PCAF gene is rs17006625A > G polymorphism in the nucleotide sequence of SEQ ID NO: 1, and the marker for diagnosing and predicting the prognosis of non-small cell lung cancer. delete delete The method according to claim 1,
Wherein said non-small cell lung cancer is a surgically resected lung cancer tissue or lung cancer cell. A kit for predicting viability of a non-small cell lung cancer patient comprising the composition of claim 1 or 2. A microarray for predicting the survival prognosis of patients with non-small cell lung cancer comprising the composition of claim 1 or 2. A method of providing information for predicting the survival prognosis of patients with non-small cell lung cancer, comprising identifying a rs17006625A> G polymorphism of the PCAF gene from nucleic acids extracted from patients with non-small cell lung cancer. 9. The method of claim 8,
Wherein the genotype of the rs17006625 polymorphism of the PCAF gene is GG, the survival prognosis is determined to be low. delete

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