KR101378540B1 - BRCA1 Haplotype Markers Associated with Survival of Non-small Cell Lung Cancer Patient and Used Thereof - Google Patents

Abstract

 The present invention provides a kit for predicting survival of non-small cell lung cancer patients treated with a platinum-containing anticancer agent comprising a haplotype consisting of single nucleotide polymorphisms (SNPs) and a method for detecting survival markers of non-small cell lung cancer patients. It is about. Prediction method using the kit of the present invention can easily and efficiently predict the survival time of patients with non-small cell lung cancer treated with platinum-containing anticancer agent, and the dose of drug to the patient using the survival time predicted by the method Effectively used to improve treatment methods, such as modulating or combining other drugs, can provide optimized treatment.

Non-small cell lung cancer, haplotype, mononucleotide polymorphism, platinum containing anticancer agent, survival prediction

Description

JPRC1 Haplotype Markers Associated with Survival of Non-small Cell Lung Cancer Patient and Used Thereof}

The present invention provides a kit for predicting survival of non-small cell lung cancer patients treated with a platinum-containing anticancer agent comprising a haplotype consisting of single nucleotide polymorphisms (SNPs) and a method for detecting survival markers of non-small cell lung cancer patients. It is about.

Lung cancer is the most common cause of cancer-related deaths in all countries. However, the survival of patients with non-small cell lung cancer (NSCLC) is slowly improving. Currently, 15% of non-small cell lung cancer patients survive for 5 years.

Despite the introduction of new drugs targeting specific molecules, cytotoxic chemotheraphy is still a major treatment for advanced non-small cell lung cancer. In cytotoxic chemotherapy, platinum containing platinum plays an essential role as one of the doublets.

Effective pharmacogenomics for lung cancer relies on identifying patients with non-small cell lung cancer that may benefit from DNA damaging agents, such as platinum. DNA repair pathway genes are of interest as potential biomarkers because individual variations in DNA repair capacity can affect the effects of DNA damaging agents. Genes such as ERCC1, RRM1, ERCC2 and XRCC1 involved in DNA repair have been concentrated as potential biomarkers for predicting the effects of platinum containing chemotherapy in non-small cell lung cancer.

BRCA1 plays an important role in DNA repair through transcriptional linked nucleotide excision repair. BRCA1 was originally known to be involved in the regulation of mitotic spindle assembly, as well as other repair processes such as top recombination repair, non-homologous end joining and mismatch repair.

However, it is not known that the germ-line variation of BRCA1 is associated with the survival of lung cancer patients.

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.

The present inventors conducted an in-depth comparison of SNPs of non-small cell lung cancer patients to develop genetic markers that can easily determine the survival time of non-small cell lung cancer patients treated with platinum-containing anticancer drugs. Was identified only in patients with NSCLC treated with SMC, and the haplotype consisting of specific combinations of these SNPs was able to determine the survival time of patients with NSCLC with very high accuracy. The invention was completed.

Accordingly, it is an object of the present invention to provide a kit for predicting survival of non-small cell lung cancer patients treated with a platinum-containing anticancer agent comprising a haplotype consisting of single nucleotide polymorphisms (SNPs).

Another object of the present invention is to provide a method for detecting a survival marker of a non-small cell lung cancer patient.

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

According to one aspect of the present invention, the present invention provides a survival time of a non-small cell lung cancer patient treated with a platinum-containing anticancer agent comprising a haplotype consisting of the following single nucleotide polymorphisms (SNPs). Provide a kit to predict:

(a) 8-100 contiguous nucleotide sequences, or complementary nucleotide sequences thereof, comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 1;

(b) 8-100 contiguous nucleotide sequences, or complementary nucleotide sequences thereof, comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 2;

(c) 8-100 contiguous nucleotide sequences or complementary nucleotide sequences thereof comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 3; And

(d) 8-100 contiguous nucleotide sequences, or complementary nucleotide sequences thereof, comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 4.

The present inventors conducted an in-depth comparison of SNPs of non-small cell lung cancer patients to develop genetic markers that can easily determine the survival time of non-small cell lung cancer patients treated with platinum-containing anticancer drugs. Was identified only in patients with NSCLC treated with SMC, and the haplotype consisting of specific combinations of these SNPs was able to determine the survival time of patients with NSCLC with very high accuracy. The invention was completed.

The SNP marker of the present invention is applied to non-small cell lung cancer patients, most preferably to non-small cell lung cancer patients treated with platinum-containing anticancer agents.

As used herein, the term “non-small cell lung cancer (NSCLC)” is used in the sense known to those skilled in the art working on lung cancer. For example, non-small cell lung cancer is a malignant cell that develops from epithelial cells of the lung, and under the microscope the size of the tumor cells observed by an observer, such as a histopathologist, and It is classified according to shape. Non-small cell lung cancer includes three major subtypes: squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma.

As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term "haplotype" refers to a series of SNPs located on one chromosome during meiosis in germ cells, which can be divided into several bundles, depending on the associations that are inherited together. This haplotype is a combination of several SNPs present on the same chromosome and shows a pattern of a certain SNP.

According to a preferred embodiment of the invention, the present invention is the 101 nucleotide r of SEQ ID NO: 1 is A or G, the 101 nucleotide m of SEQ ID NO: 2 is A or C, the sequence of the third sequence The 101st nucleotide y is C or T, and the 101st nucleotide m of the SEQ ID NO: 4 sequence is C or A.

SNP variations in polynucleotides comprising nucleotides exhibiting the 101 st mononucleotide polymorphism of SEQ ID NO: 1 sequence are also described herein as "S1613G (A> G)". In addition, SNP mutations in polynucleotides containing nucleotides exhibiting the 101 st mononucleotide polymorphism of the SEQ ID NO: 2 sequence are also described as "IVS13-1893 (A> C)", and the 101 st single of the SEQ ID NO 3 sequence SNP mutations in polynucleotides comprising nucleotides exhibiting nucleotide polymorphisms are also described as "IVS12-1207 (C> T)". In addition, SNP mutations in polynucleotides containing nucleotides representing the 101 st mononucleotide polymorphism of SEQ ID NO: 4 are also described as "IVS12 + 112 (C> A)".

In these four monobasic polymorphisms (SNPs), S1613G (A> G) is located at exon 16 in the human BRCA1 gene, IVS13-1893 (A> C) is at intron 13, IVS12-1207 (C> T) and IVS12 + 112 (C> A) is located at intron 12 and strongly correlates with the survival of non-small cell lung cancer patients treated with platinum-containing anticancer agents.

According to a preferred embodiment of the present invention, the haplotype included in the present invention has a single nucleotide polymorphism located in the 101 nucleotide of the first sequence of SEQ ID NO: A, a nucleotide located in the 101 nucleotide of the second sequence of SEQ ID NO: The polymorphism is A, the single nucleotide polymorphism located at the 101 nucleotide of SEQ ID NO: 3 sequence is C, the single nucleotide polymorphism located at the 101 nucleotide of the SEQ ID NO: 4 sequence is C.

That is, when two copies of a haplotype of the SNP positions determined for the SNP positions of the polynucleotides of SEQ ID NOS: 1 to 4 are present in the individual, the haplotype is 0 or 1 Determining that the survival time is short compared to the subject, or if the haplotype is present in the subject with zero or one copy, determining that the haplotype is longer than the subject with two copies. The survival period can be determined, for example, for individuals with two copies of the AACC haplotype to survive 6-7 months shorter than for individuals with zero or one copy.

Thus, for individuals with two copies of the AACC haplotype, the survival is expected to be short, and in addition to the chemotherapy with platinum drugs, it is possible to consider using other secondary drugs or treatment methods. In addition, for individuals with two copies of the AACC haplotype, it is possible to consider using a dosing regimen, eg, adjusting the dose of the drug, in order to increase the efficacy of the drug as compared to the individual having zero or one copy.

The term "Linkage Disequilibrium" as used herein refers to a non-random association of alleles in two or more loci that are not necessarily on the same chromosome in population genetics. it means. That is, associative equilibrium is a measure of binding between alleles at different positions, and if each gene is combined completely independently, the gene set will probably maintain a linkage equilibrium, but some alleles are found together. There are many examples, that is, they are not randomly mixed, and these alleles are in an associated equilibrium state. This associated non-equilibrium state is interpreted as a result of natural selection when alleles are located close together or when alleles are clustered together.

The present invention relates to base variants of the respective SNP positions in the SEQ ID NO: 1 to 4 sequences, but when such SNP base mutations are found in double stranded genomic DNA (gDNA), complementary to the nucleotide sequences described above It is also interpreted to include polynucleotide sequences. Thus, the base at the SNP position in the complementary polynucleotide sequence is a complementary base.

According to a preferred embodiment of the invention, the 8-100 consecutive nucleotides are primers for nucleic acid amplification or probes for detecting the nucleotide sequence of the nucleic acid.

As used herein, the term “nucleic acid” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are naturally occurring nucleotides, as well as analogs in which sugar or base sites are modified. analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

In the present invention, the nucleic acid can be obtained from various sources of non-pulmonary cancer patients, for example, from muscle, epidermis, blood, bone, organs, and most preferably from muscle or blood.

If the starting material in the present invention is gDNA, isolation of gDNA can be carried out according to conventional methods known in the art (Rogers & Bendich (1994)). If the starting material is mRNA, total RNA is isolated and performed by conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press ( Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized by cDNA using reverse transcriptase. Since the total RNA is isolated from human cells, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primer and reverse transcriptase using this sequence characteristic (see 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) )).

In the present invention, the method for identifying the base type of the single nucleotide polymorphism (SNP) in the separated nucleic acid molecule may be carried out by applying various methods known in the art. For example, techniques applicable to the present invention include single base primer extension assays, fluorescence in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded Conformation assays (SSCA, Orita et al., PNAS, USA 86: 2776 (1989)), RNase protection assays (Finkelstein et al., Genomics, 7: 167 (1990)), dot blot analysis, denaturation gradient gel electrophoresis (DGGE, Wartell et al., Nucl. Acids Res., 18: 2699 (1990)), methods using proteins that recognize nucleotide mismatches (e.g., mutS protein of E. coli) (Modrich, Ann. Rev. Genet , 25: 229-253 (1991)), and allele-specific PCR.

Sequence changes result in differences in base bonds within single-stranded molecules, resulting in different bands of mobility, which SSCA detects. DGGE analysis uses a denaturing gradient gel to detect sequences that represent wild type sequences and other mobility. Other techniques generally employ probes or primers that are complementary to sequences comprising the SNPs of the invention. For example, in an RNase protection assay, a riboprobe complementary to a sequence comprising a SNP of the present invention is used. The riboprobe is hybridized with DNA or mRNA isolated from a plant, and then cleaved with an RNase A enzyme capable of detecting a mismatch. If there is a mismatch and RNase A recognizes, a smaller band is observed.

In assays using hybridization signals, probes complementary to the sequences comprising the SNPs of the invention are used. In this technique, hybridization signals of probes and target sequences are detected to directly determine whether SNP variants are present.

As used herein, the term “probe” refers to a linear oligomer having naturally occurring or modified monomers or bonds, including deoxyribonucleotides and ribonucleotides that can hybridize to a particular nucleotide sequence. Preferably, the probe is single stranded for maximum efficiency in hybridization. The probe is preferably deoxyribonucleotide.

As the probe used in the present invention, a sequence perfectly complementary to the sequence including the SNP may be used, but a sequence complementarily complementary to a range that does not prevent specific hybridization may be used. It may be. Preferably, the probe used in the present invention hybridizes to a sequence comprising 8-100 contiguous nucleotides comprising the 101st nucleotide of SEQ ID NO: 1, 2, 3 and 4 of the SNP nucleotides. Sequences that can be included. More preferably, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If the part is not hybridized, such a duplex can be disassembled under stringent conditions. Conditions suitable for hybridization include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). ≪ / RTI > Stringent conditions used for hybridization can be determined by adjusting the temperature, ionic strength (buffer concentration), the presence of compounds such as organic solvents, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

When SNP is applied to a gene amplification method, it is especially called single nucleotide amplified polymophism (SNAP).

According to a preferred embodiment of the present invention, such gene amplification is a primer designed to amplify a polynucleotide comprising the 101st nucleotide of SNP nucleotides SEQ ID NO: 1, 2, 3, and 4 Basic pairs are used by default.

A “primer” in the context of the present invention is a single strand of oligonucleotide, suitable conditions (the presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerases), suitable temperatures and suitable It is meant to act as an initiation point that can initiate template-directed DNA synthesis under a buffer.

The suitable length of the primer is determined by the characteristics of the primer to be used, but is usually 15 to 30 bp in length. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

Amplification techniques that can be used in the methods of the invention include PCR amplification (Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822)), ligase chain reactions (LCR, Wu, DY) et al., Genomics 4: 560 (1989)), polymerase ligase chain reaction (Barany, PCR Methods and Applic., 1: 5-16 (1991)), Gap-LCR (WO 90/01069), repair chain Reactions (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86: 1173 (1989)) and NASBA (US Pat. No. 5,130,238). Most preferably by PCR amplification step.

Where amplification is applied, it is important to design suitable primers to identify the SNP bases of the present invention.

The conditions of the amplification reaction by PCR and the reagents and enzymes used can be those conventionally known in the art.

According to another aspect of the invention, the invention provides a method of (a) sequencing the first sequence in a biological sample of a lung cancer patient to provide information necessary for predicting survival of non-small cell lung cancer patients treated with platinum-containing anticancer agents. Single nucleotide polymorphism (SNP) located at the 101st nucleotide; (b) single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 2; (c) a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 3; And (d) a non-small cell lung cancer patient treated with a platinum-containing anticancer agent by detecting haplotypes with a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 4. It provides a method for detecting a survival marker of.

Since the method of the present invention includes the SNP, the contents in common between the two are omitted in order to avoid excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the 101 nucleotide r of the sequence 1 st sequence is A or G, the 101 nucleotide m of the sequence 2 sequence is A or C, the 101 st of the sequence 3 sequence Nucleotide y is C or T, and the 101th nucleotide m of the sequence 4th sequence is C or A, most preferably a single nucleotide polymorphism located at the 101st nucleotide of the sequence 1st sequence is A, The single nucleotide polymorphism located at 101 nucleotide of the second sequence is A, the single nucleotide polymorphism located at 101 nucleotide of the sequence 3rd sequence is C and the single nucleotide polymorphism is located at 101 nucleotide of the 4th sequence It is C.

According to a preferred embodiment of the present invention, the method comprises said doubling if a haplotype of A, A, C and C nucleotides of the SNP positions of SEQ ID NOS 1 to 4 are present in the patient sample Determining that survival is short compared to patients with zero or one copy of the body, or having zero or one copy of haplotype in the patient, or longer survival than two copies of the haplotype. will be.

According to a preferred embodiment of the present invention, the non-small cell lung cancer is squamous cell cancer, adenocarcinoma or large cell cancer.

According to a preferred embodiment of the present invention, the patient is treated two or more times with a platinum-containing anticancer agent or a combination of a platinum-containing anticancer agent and a platinum-free anticancer agent.

Platinum-containing anticancer drugs are drugs that contain platinum molecules in their chemical structure and bind to DNA of cancer cells to inhibit replication, thereby causing cancer cytotoxicity. Platinum-containing anticancer agent herein includes anticancer agents including various platinum known in the art, preferably cis-platin, carboplatin, oxaliplatin, paraplatin And one or more anticancer agent selected from the group consisting of nedaplatin (nedaplatin).

Platinum-containing anticancer drugs are the most widely used anticancer drugs among the widespread anticancer drugs currently used, but are widely used for the treatment of cancer, but due to problems such as resistance to platinum anticancer drugs in various types of cancer cells, they do not contain platinum. An anticancer drug is used in combination. The platinum-free anticancer agent herein includes an anticancer agent that does not include various platinum known in the art, and is preferably selected from the group consisting of gemcitabine, taxane and vinca alkaloids. It is one or two or more anticancer agents. The term "taxne" refers to a mitotic inhibitor that disrupts the function of the microtube. The taxanes include paclitaxel, docetaxel, lorataxel, ortataxel and tesetaxel. The "vinca alkaloid" refers to anti-mitotic and anti-microtubule preparations. The vinca alkaloids include vinblastine, vincristine, vindesine and vinoreline.

According to a preferred embodiment of the present invention, the lung cancer patient is a lung cancer patient in stage IIIA, IIIB or IV.

Stages IIIA, IIIB and IV are well known in the art as part of the lung cancer classification system proposed by Clifton Mountain. The Mountain classification system assigns stages based on the similarity of treatment and prognosis. Stage IIIA corresponds to T1N2MO, T2N2MO, T3N1MO or T3N2MO of the TMN stage system, stage IIIB corresponds to any T N3MO or T4 any N MO of the TMN stage system, and stage IV corresponds to any T any N M1 Corresponding. The TMN system is well known to those skilled in the art and the T, N and M are as known in the TMN system. In the TMN system, T represents the primary tumour, T1 is surrounded by the lungs or the visceral pleura and is the largest, without bronchoscopic invasion into the main bronchus. Has a primary tumor of less than 3 cm in dimension, T2 has a tumor of 3 cm or more in its largest dimension, a tumor that extends into the main bronchus but is more than 2 cm away from the carina, and obstructive pneumonitis , One of the tumors that is not related to the entire lung, and T3 is a tumor with involvement of the chest wall, diaphragm, mediastinal pleura, or parietal pericardium , Within 2 cm of the keel, extending into the main bronchus but not related to the keel, and one of the tumors with obstructive pneumonia of the entire lung, T4 is a tumor with an invasion of the mediastinum, heart, great vessels, trachea, esophagus, vertebra, or keel, separate tumors in the same lobe One of a tumor with nodule and a tumor with malignant pleural effusion. N represents the lymph node, N1 metastasizes to the ipsilateral peribronchial or ipsilateral hilar lymph nodes, N2 metastases to the same choroidal or subcarinal lymph nodes. N3 is supraclavicular lymph node. Metastases to one of the same scalene lymph nodes, the opposite one. M indicates distant metastasis, M0 indicates no distant metastasis, and M1 indicates distant metastasis.

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

(Iii) The present invention provides a kit for predicting survival of non-small cell lung cancer patients treated with a platinum-containing anticancer agent comprising a haplotype consisting of a single nucleotide polymorphism (SNP) and a survival marker of a non-small cell lung cancer patient. Provide a way to.

(Ii) The prediction method using the kit of the present invention can easily and efficiently predict the survival time of patients with non-small cell lung cancer treated with platinum-containing anticancer agent, and uses the survival time predicted by the method for the patient. Effective treatment can be used to improve the treatment method, such as adjusting the dose of a drug or combining other drugs, thereby providing an optimized treatment.

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 Experiments

Effect of BRCA1 Haplotype on the Survival of Non-small Cell Lung Cancer Patients Treated with Platinum-containing Chemotherapy

In this Example, the genomic-line variation of BRAC1 was confirmed on the survival of non-small cell lung cancer (NSCLC) patients treated with platinum-containing anticancer therapies. To this end, for 300 Korean NSCLC patients in stages IIIA (16%), IIIB (31%) and IV (53%), whether four BRCA1 SNPs and their haplotypes are associated with treatment outcomes Was evaluated. As a result, it was confirmed that the AACC haplotype of the BRCA1 gene is associated with the survival time of NSCLC patients treated with platinum-containing anticancer drugs.

2. Selection of research groups and acquisition of clinical information

Lung Cancer Cohort of Inha University Hospital (Incheon, Korea) constructed a database containing clinical information and corresponding peripheral-blood DNA sequence information. The clinical information was obtained after interviewing all lung cancer patients when a well-trained research nurse was diagnosed with lung cancer. Uniform data sheet with information on treatment, tumor response, follow-up and survival, smoking habits, Eastern Cooperative Oncology Group performance status and weight loss Was collected from the group using. In order to increase the quality of this information, physicians (JSR) and research nurses (HJK) have two weeks to review the results of electronic charts and radiologic examinations or laboratory studies for each patient. Every review. If the patient moves to a primary or secondary hospital, or if the patient leaves the hospital to stay at home for conservative or terminal care, call or mail the patient or his or her relatives to check the patient's condition. Contact was made through.

From more than 1,000 NSCLC patients diagnosed between March 2000 and May 2006, two or more treatments with platinum-based anticancer drugs as first-line treatment, 300 patients were selected, with complete follow-up, the patient's peripheral blood lymphocytes available for analysis, and with advanced stage disease.

The 300 selected patients were informed of the study purpose and agreed in writing. The study was approved by the Institutional Review Board of Inha University Hospital.

3. Selection of tagging polymorphism

The BRCA1 gene spans about 81.2 kb and contains 24 exons. All polymorphisms between -1,100 nucleotides upstream from the initiation codon (ATG) and the 3 'untranslated region of the BRCA1 gene were investigated using the following two data systems: the International HapMap Project and the Japanese Single-Nucleotide. Polymorphisms.

35 polymorphs were selected with minor allele frequency of more than 10% in Asian ethnic populations. Of these polymorphisms, four tSNPs were selected using the criterion that the value of the correlation coefficient r 2 was greater than 0.9. Four tSNPs were selected with the following priorities. (1) nonsynonymous coding polymorphism; (2) synonymous coding polymorphism; (3) polymorphism near the start codon; (4) intronic polymorphism near an exon; And (5) downstream.

Finally, IVS12 + 112 (C>A; rs2070833 ), IVS12-1207 (C>T; rs8067269 ), IVS13-1893A / C ( rs8176199 ), and S1613G ( rs1799966 ) were identified as tSNPs in this example. . Table 1 shows information for four tSNPs. Table 1 contains the primers and allele frequencies used for amplification of tSNPs.

In Table 1, SNPs are single-nucleotide polymorphisms; HWE stands for Hardy-Weinberg equilibrium.

4. Genetic Analysis

Genotyping analysis for the four SNPs shown in Table 1 was performed using the GenomeLab SNPstream Genotyping System (ultra-high throughput [UHT]; Beckman Coulter, Fullerton, CA), according to the manufacturer's protocol.

Polymerase chain reaction (PCR) amplification was performed in a PTC-225 Peltier Thermal Cycler (MJ Research, Waltham, Mass.) Using Taq Gold DNA polymerase. The sequences of the PCR and extension primers are shown in Table 1. The S1613G genotype was analyzed using a single-base primer extension assay using the SNaPShot assay kit (Applied Biosystems [ABI], Foster City, Calif.) According to the manufacturer's protocol. PCR product sequences were analyzed using electrophoresis on an ABI Prism 3730 DNA analyzer. The results were analyzed using Gene Mapper software (ABI).

5. Survival Measurement

Survival was measured from the day of initiation of treatment with platinum containing anticancer agent. The date of death was obtained primarily by contacting the patient's relatives by phone or mail or by reviewing the electronic chart. Seven of the patients could not be contacted because they were not followed after being discharged from the hospital. Information about them was collected from the National Statistical Office or the National Police Agency (Seoul, Korea).

6. Statistical Method

The χ ² test for heterogeneity was used to compare the distribution of clinical variables or genotype frequencies, and Mann-Whitney U. The test was used for continuous variables. Whether the allele frequency of the four tSNPs is in the Hardy-Weinberg equilibrium is determined by Haploview v. The determination was made using a 4.0 software package and the values of Lewontin's coefficient, D 'and r 2 were estimated.

The association of genotype or clinical variables with overall survival was estimated using the Kaplan-Meier method and log-rank testing for univariate analysis. . Adjusted risk ratio and 95% confidence interval (CI) for potential confounders are determined using the Cox proportional hazards model for multivariate analysis. It was.

The association between the haplotype copy number and overall survival was determined using a recessive model. All analyzes were performed using the SAS software package (version 9.1.3; SAS Institute, Cary, NC).

Experiment result

1. Patient and Treatment

Demographic clinical information for 300 patients used in this example is shown in Table 2. The median age of the 300 patients was 63 years (28-89 years), of which 238 (79%) were male. Most of the patients (223 out of 300, 74%) were 1 or greater in the ECOG Behavioral Status (Eastern Cooperative Oncology Group performance status). At the time of diagnosis, 16% of patients had stage IIIA lung cancer, 31% had stage IIIB lung cancer, and 53% had stage IV lung cancer.

For histologic cell type, 46.3% of patients were squamous cell carcinomas and 45.7% were adenocarcinoma. As first-line chemotheraphy, 159 patients (53%) received taxane doublets and 139 patients (46%) received gemcitabine doublets. Of the 163 (54%) who received second-line chemotherapy, 91% were treated with platinum doublets and single cytotoxic agents. Here, taxane doublets represent a combination treatment of taxanes (paclitaxel, docetaxel) and platinum containing anticancer agents, and gemcitabine doublets represent a combination treatment of gemcitabine and platinum containing anticancer agents. Platinum doublets represent combination treatments between platinum containing anticancer agents.

Radiation therapy for primary tumors was administered to 89 patients (30%). Event (death) was observed in 290 of 300 patients (90%). Tables 2a-b show the characteristics of the patients selected in this example and a copy of the AACC haplotype of BRCA1.

In Table 2a-b, EGOG represents Eastern Cooperative Oncology Group, EGFR represents epidermal growth factor receptor, and TKI represents tyrosine kinase inhibitor.

2. BRCA1 4 tags in SNP ( tagging SNP ) Genotype and Allele  frequency

Three of the tSNPs were located in introns. Another tSNP located at exon 16 was a nonsynonymous polymorphism that caused an amino acid change, ie, a change in serine to glycine. Hardy-Wineberg equilibrium was observed for 4 tSNPs. The frequency of variant alleles is as follows: 30% for S1613G, 17% for IVS13-1893 (A> C), 40% for IVS12-1207 (C> T), and IVS12 + 112 27% for (C> A). Each tSNP, gender and ECOG behavior status (0 to 1 set) ≥ 2), histological cell type (squamous cell carcinoma vs. adenocarcinoma), smoking habits (never smoker versus ever smoker), stage (III versus IV), tumor response (complete or incomplete relapse versus stable or progressive disease), primary treatment (gemcitabine) Large taxane), secondary treatment (yes versus no), or sequential radiation therapy (yes versus no data).

3. Association equilibrium ( linkage disequilibrium )and BRCA1 Haplotype

High linkage disequilibrium (LD) was observed for all four tSNPs with a D value of 1, but the r ² value was moderate (range: 0.08 to 0.664). Haploview v. Using the 4.0 software package, the haplotypes of BRCA1 were constructed in the following order: S1613G, IVS13-1893 (A> C), IVS12-1207 (C> T) and IVS12 + 112 (C>A); Six of these haplotypes were identified. Five of the six haplotypes were the most common haplotypes, AACC, AACA, GCTC, GATC and AATC, which together accounted for 99.9% of all haplotypes. AACC haplotype was most frequently observed with a frequency of 32.1%.

4. tSNP And overall survival

The median survival time (MST) of 300 patients was 13.0 months. No association was observed between tSNP and patient survival (data not shown).

5. Clinical variables or BRCA1 of Haplotype and  Association of overall survival

When analyzing the association between clinical variables and the patient's overall survival, weight loss (≥5%), ECOG behavior status (≥2), stage (IV), secondary treatment (no) or radiotherapy (no) log According to the rank test and the Cox proportional hazards model, it was significantly associated with shorter survival (Table 3). Other clinical variables investigated (gender, smoking habits, histological cell types, and primary treatment) were not prognostic factors in the context of this example.

292 patients were divided into two groups according to the number of copies of alleles of each BRCA1 haplotype. One group with 0 or 1 copy and the other group with 2 copies. The recessive model was used to statistically analyze these two groups and to assess the association between the copy number and overall survival of the five BRCA1 haplotypes. As a result, the association between any of the four low frequency haplotypes (AACA, GCTC, GATC, and AATC) and survival time could not be demonstrated (data not shown). However, it was demonstrated that patients with two copies of AACC, wild type haplotype, had significantly shorter survival compared to patients with zero or one copy (MST, 8.5 months vs. 14.6 months); Log-rank P = 0.0066; Table 3). After adjusting for the confounding variables identified by the monovariate analysis, the Cox model found that patients with 2 copies had a 2 times higher risk of death compared to patients with 0 or 1 copy of AACC haplotype. (Hazard ratio [HR] = 2.097; 95% CI, 1.339 to 3.284). Between two groups separated by AACC haplotype or clinical variable, except according to the copy number of the AACC haplotype. No association was observed (Table 1).

6. BRCA1 of AACC Haplotype and  Overall survival: subgroup analysis

We analyzed the effect of copy number of wild type haplotype (AACC) on survival according to cell type, smoking habits, and chemotherapy (chemotheraphy regimen).

In patients with squamous cell carcinoma, the MST of patients with 2 copies was 6.8 months and less than half the MST of patients with 0 or 1 copy (log-rank P = 3.6 × 10 ⁻⁵ ). In the Cox model, patients with 2 copies showed an increased risk of death (HR = 3.20; 95% CI, 1.74 to 5.97). In contrast, no association was demonstrated in patients with adenocarcinoma (log-rank P = 0.6774; HR = 0.919; 95% CI, 0.429 to 1.968). Analyzes of copy number effects by smoking habit or gender showed that smokers or men with two copies had significantly shorter survival (log-rank P = 0.0016; HR = 2.01; 95% for smokers). CI, 1.248-3.240; log-rank P = 0.0070 for men; HR = 1.96; 95% CI, 1.217-3.187). For chemotherapy, patients with 2 copies had significantly shorter survival in taxane / cisplatin-treated patients, but did not show this effect in gemcitabine / cisplatin-treated patients (log-rank P for taxane / cisplatin). = 0.0082; HR = 2.053; 95% CI, 1.123 to 3.752; log-rank log-rank P = 0.3009 for gemcitabine / cisplatin; HR = 1.606; 95% CI, 0.763 to 3.382).

In Table 3, MST represents median survival time, ECOG is Eastern Cooperative Oncology Group, and P and D are paclitaxel / docetaxel. * Is estimated from the Cox proportional hazard's model, adjusted for weight loss, ECOG behavior status, stage, secondary treatment, and radiation treatment.

The results of the above examples demonstrate that the number of copies of the haplotype AACC is associated with the survival of patients treated with platinum containing anticancer agents. Based on this association, it is possible to distinguish between patients with two copies of the haplotype AACC and those who do not, to provide optimized treatment.

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.

<110> INHA Industry Partnership Institute <120> BRCA1 Haplotype Markers Associated with Survival of Non-small          Cell Lung Cancer Patient and Used Thereof <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 201 <212> DNA <213> Homo sapiens <400> 1 ttctgaagac agagccccag agtcagctcg tgttggcaac ataccatctt caacctctgc 60 attgaaagtt ccccaattga aagttgcaga atctgcccag rgtccagctg ctgctcatac 120 tactgatact gctgggtata atgcaatgga agaaagtgtg agcagggaga agccagaatt 180 gacagcttca acagaaaggg t 201 <210> 2 <211> 201 <212> DNA <213> Homo sapiens <400> 2 ttctacctat atttatggct tttagctttt ctaataaaag ctcaaaatga attacagtca 60 tcagtgactt tttaatgaat agaagacttt tgcaattttt mactatttgt ttttacttat 120 taaatatttc cgccttggcc aggcatggtg gctcacgcct ataatcccag cactgtgaga 180 tgccaaggca ggaggatcac t 201 <210> 3 <211> 201 <212> DNA <213> Homo sapiens <400> 3 agagccctga gaaagcctgt aaggatagtt actgttttta aaataatgag ttctttttgc 60 ttatgggctc ctgttgttta ttggtccatt tcaaagaaga ytgtgctaag tccaagtatt 120 tgataaacaa agaatttagg tatgtaagga gttttccaaa atatccttct taagaattta 180 ttttatttat ttattttttt t 201 <210> 4 <211> 201 <212> DNA <213> Homo sapiens <400> 4 gtgtgtgtgt gcacatgcgt gtgtgtggtg tcctttgcat tcagtagtat gtatcccaca 60 ttcttaggtt tgctgacatc atctctttga attaatggca maattgtttg tggttcattg 120 tctccttaaa ttagactgta agcaccttga tggaactcat actacctttt atttcacaca 180 cacgcacacg cgcacacaca g 201 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs1799966 amplification <400> 5 aacataccat cttcaacctc tgc 23 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs1799966 amplification <400> 6 aattctggct tctccctgc 19 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Extension primer for rs1799966 allele <400> 7 rttgaaagtt gcagaatctg cccag 25 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs8176199 amplification <400> 8 tgaattacag tcatcagtga cttttt 26 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs8176199 amplification <400> 9 gcctggccaa ggcggaaata t 21 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Extension primer for rs8176199 allele <400> 10 acgcacgtcc acggtgattt tatttaataa gtaaaaacaa atagt 45 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs8067269 amplification <400> 11 tttggaaaac tccttacata cctaa 25 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs8067269 amplification <400> 12 ctgtaaggat agttactgtt tttaaaataa tg 32 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Extension primer for rs8067269 allele <400> 13 ggatggcgtt ccgtcctatt ttatcaaata cttggactta gcaca 45 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs2070833 amplification <400> 14 tcctttgcat tcagtagtat gtatc 25 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs2070833 amplification <400> 15 taaaaggtag tatgagttcc atca 24 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Extension primer for rs2070833 allele <400> 16 ggctatgatt cgcaatgctt aggagacaat gaaccacaaa caatt 45

Claims (13)

Kit for predicting survival of non-small cell lung cancer patients treated with a platinum-containing anticancer agent comprising a haplotype consisting of the following single nucleotide polymorphisms (SNPs): (a) 8-100 contiguous nucleotide sequences comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 1, or a complementary nucleotide sequence thereof, wherein said SEQ ID NO: 1 The 101 nucleotide r of is A or G; (b) 8-100 contiguous nucleotide sequences comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 2, or a complementary nucleotide sequence thereof, wherein the second SEQ ID NO: The 101 nucleotide m of is A or C; (c) 8-100 contiguous nucleotide sequences comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 3, or a complementary nucleotide sequence thereof, wherein said SEQ ID NO: 3 The 101st nucleotide y of is C or T; And (d) 8-100 contiguous nucleotide sequences comprising a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 4, or a complementary nucleotide sequence thereof, wherein said SEQ ID NO: 4 The 101 nucleotide m of is C or A. delete The method of claim 1, wherein the haplotype is a single nucleotide polymorphism located in the 101 nucleotide of SEQ ID NO: 1 sequence, A single nucleotide polymorphism located in the 101 nucleotide of the second sequence SEQ ID NO: A, SEQ ID NO: 3 And wherein the single nucleotide polymorphism located at the 101 nucleotide of the sequence is C, the single nucleotide polymorphism located at the 101 nucleotide of the SEQ ID NO: 4 sequence is C. The kit of claim 1, wherein the 8-100 contiguous nucleotides are primers for nucleic acid amplification or probes for detecting the nucleotide sequence of the nucleic acid. (A) A single nucleotide polymorphism located at the 101st nucleotide of sequence 1st sequence in a biological sample of lung cancer patient to provide information necessary to predict survival of non-small cell lung cancer patients treated with platinum-containing anticancer drugs. nucleotide polymorphism (SNP); (b) single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 2; (c) a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 3; And (d) non-small cell lung cancer patients treated with platinum-containing anticancer agents by detecting haplotypes with a single nucleotide polymorphism (SNP) located at the 101st nucleotide of SEQ ID NO: 4. A method of detecting a survival marker, wherein the 101 nucleotide r of the first sequence of the sequence is A or G, the 101 nucleotide m of the second sequence of the sequence is A or C, the 101st of the third sequence of the sequence Wherein nucleotide y is C or T, and the 101th nucleotide m of said SEQ ID NO: 4 sequence is C or A. delete The method according to claim 5, wherein the haplotype is a single nucleotide polymorphism located in the 101 nucleotide of SEQ ID NO: 1 sequence, A single nucleotide polymorphism located in the 101 nucleotide of the second sequence SEQ ID NO: A, SEQ ID NO: 3 Wherein the single nucleotide polymorphism located at the 101 nucleotide of the sequence is C, the single nucleotide polymorphism located at the 101 nucleotide of the SEQ ID NO: 4 sequence is C. 6. The method according to claim 5, wherein the haplotype is 0 when there are two copies of a haplotype in the patient sample wherein the nucleotides of the SNP positions of SEQ ID NOS 1 to 4 are each A, A, C and C, respectively. Or a survival time is shorter than that of a patient having one copy, or when the haplotype is 0 or 1 copy present in the patient, the survival time is determined to be longer than that of a patient having 2 copies of the haplotype. How to. 6. The method of claim 5, wherein the non-small cell lung cancer is squamous cell cancer, adenocarcinoma or large cell cancer. The method of claim 5, wherein the patient has been treated with a platinum-containing anticancer agent or a combination of a platinum-containing anticancer agent and a platinum-free anticancer agent at least twice. 6. The method of claim 5, wherein the platinum containing anticancer agent is one or more anticancer agents selected from the group consisting of cisplatin, carboplatin, oxaliplatin and nedaplatin. The method of claim 10, wherein the platinum-free anticancer agent is one or more anticancer agents selected from the group consisting of gemcitabine, taxane and vinca alkaloids. 6. The method of claim 5, wherein the lung cancer patient is a lung cancer patient of stage IIIA, IIIB or IV.