KR20220122219A - Diagnostic methods for prognosis of small-cell lung cancer using MFF SNP - Google Patents

Abstract

The present invention relates to a method for diagnosing or predicting the survival prognosis of small cell lung cancer using rs7559435 single nucleotide polymorphism present in a promoter region of MFF gene, and a use thereof. The technology for predicting the survival prognosis of small cell lung cancer according to the present invention enables easy and accurate prediction of the prognosis of patients with small cell lung cancer, and can also be applied to methods for the selection and evaluation of appropriate therapies. Furthermore, the survival rate of small cell lung cancer patients can be increased through new targeted therapies for small cell lung cancer.

Description

Method for prognostic diagnosis of small-cell lung cancer using single nucleotide polymorphism of MFF {Diagnostic methods for prognosis of small-cell lung cancer using MFF SNP}

The present invention relates to a method for diagnosing the prognosis of small cell lung cancer using a single nucleotide polymorphism of MFF. Specifically, the present invention relates to a method for diagnosing or predicting the survival prognosis of small cell lung cancer using the rs7559435 single nucleotide polymorphism present in the promoter region of the MFF gene. to methods and uses thereof.

Globally, lung cancer causes 1,600,000 new cases each year, of which 1,400,000 die. It accounts for 13% of all cancer cases and 18% of cancer-related deaths. In 2010, lung cancer deaths increased to 1.5 million, accounting for 19% of all cancer-related deaths.

Lung cancer is divided into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Non-small cell lung cancer, for which surgery is the best treatment option at an early stage, accounts for about 85% of all lung cancers. Small cell lung cancer is a very aggressive carcinoma, accounting for about 15% of all lung cancers. Small cell lung cancer has smaller cancer cells than non-small cell lung cancer and consists of neuroendocrine cancer cells. In particular, cancer cells grow very quickly and metastasize easily even in the early stages, which is known as a carcinoma with a poor prognosis, and the 5-year survival rate is very low between 5% and 10%. With the remarkable advances in life sciences over the past few decades, the 5-year survival rate for most cancers has increased significantly compared to 30-40 years ago, while the survival rate for small cell lung cancer remains almost the same. This is because, unlike non-small cell lung cancer, an effective treatment method for small cell lung cancer has not yet been developed.

In addition, since cancer often recurs locally or metastasizes to distant tissues and organs from the original cancer tissue, it is a disease that can be effectively treated only when a treatment method is appropriately selected according to the stage. It is very important to predict

The ideal treatment for lung cancer is to detect it early and completely remove the cancer with surgery. In addition, after the onset of lung cancer, especially small cell cancer, there is very little possibility that it can be treated through surgical operation when it is detected. Current clinical diagnostic methods cannot confirm the prognosis of early small cell lung cancer with sufficient accuracy to instruct more aggressive therapy for patients with a high risk of relapse.

Currently, the most widely used treatment method for small cell lung cancer is chemotherapy, which is the administration of complex anticancer drugs such as cisplatin and etoposide. They act on DNA to stop the growth and proliferation of cancer cells, leading to the death of cancer cells. The initial response rate of this combination anticancer drug is about 45-80%, and it responds effectively to many small cell lung cancer patients. However, since it does not act specifically against cancer cells, but also responds to normal cells, it shows many side effects to patients. Even worse, most patients develop resistance to these chemotherapy drugs within a few months. Therefore, there is no significant difference in the 5-year survival rate of small cell lung cancer patients. As such, many studies have been conducted on the mechanisms by which cancer cells become resistant to chemotherapy, mutations in transpoters that help the absorption of drugs into cells, abnormal expression of topoisomerase, increase in intercellular binding force, increase in cancer stem cells, diversity of cancer cells, etc. This is suggested as the cause. However, research on the mechanism of resistance to chemotherapy in small cell lung cancer is particularly slow, and one of the biggest reasons is that it is very difficult to obtain patient tissue before and after administration of chemotherapy. In the case of other carcinomas, it is common to remove the cancerous tissue from the patient because surgical treatment works effectively. is being done on a limited basis. This is a huge limitation for researchers in conducting research on resistance, growth, and metastasis of small cell lung cancer. Therefore, small cell lung cancer mouse models are being used for research on growth, metastasis, and resistance to small cell lung cancer, response to drug treatment, and screening for therapeutic agents.

A number of clinical trials for small cell lung cancer have been conducted similarly to other carcinomas, but most clinical trials have shown negative results. Therefore, it is expected that the clinical trials of the targeted therapeutics listed above will be conducted as soon as possible. Furthermore, screening and R&D of new complex anticancer drugs that can be effectively administered to small cell lung cancer patients are required. is in need of development.

Mitochondrial fission factor (MFF) is an outer mitochondrial membrane protein that binds to the GTPase Drp1, and is known to play a role in regulating mitochondrial division. The present inventors have completed the present invention by confirming that the single nucleotide polymorphism present in the MFF gene is closely related to the clinical outcome of SCLC patients.

It is an object of the present invention to provide a composition for a marker for the diagnosis and prediction of prognosis of small cell lung cancer including the rs7559435 polymorphism of the mitochondrial fission factor (MFF) gene.

Another object of the present invention is to provide a composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy, comprising an agent capable of detecting the rs7559435 polymorphism of the mitochondrial fission factor (MFF) gene.

Another object of the present invention is to provide a kit for predicting the survival prognosis of a patient with small cell lung cancer or a therapeutic response to chemotherapy, comprising the composition.

Another object of the present invention is to provide a microarray for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy comprising the composition.

In addition, another object of the present invention is to provide a method for providing information for the survival prognosis of a patient with small cell lung cancer or a therapeutic response to chemotherapy.

In order to achieve the above object, the present invention provides a prognosis of small cell lung cancer comprising a single nucleotide polymorphism (SNP) of rs7559435 located at the 501st nucleotide sequence of the mitochondrial fission factor (MFF) gene represented by the nucleotide sequence of SEQ ID NO: 1 A composition for a marker for diagnosis and prediction is provided.

Subsequently, the present invention provides a composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy comprising an agent capable of detecting a single nucleotide polymorphism (SNP) located in the 501st nucleotide sequence of SEQ ID NO: 1 do.

Furthermore, the present invention provides a kit or microarray for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy, comprising the composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy.

In addition, the present invention predicts the survival prognosis of small cell lung cancer patients or the prediction of treatment response to chemotherapy, including the step of confirming the rs7559435 polymorphism present in the promoter region of the mitochondrial fission factor (MFF) gene with respect to the nucleic acid extracted from the sample. It provides a way to provide information for

The technology for predicting the survival prognosis of small cell lung cancer according to the present invention can predict the patient's prognosis easily and accurately for a patient with small cell lung cancer, and can be applied to a method for selection and evaluation of an appropriate treatment, furthermore, a new method for small cell lung cancer Targeted therapy can increase the survival rate of patients with small cell lung cancer.

1 is a diagram showing ChIP-seq results for the small cell lung cancer cell line H146.
2 is a schematic diagram briefly illustrating the process of selecting candidate biomarkers for predicting the prognosis of small cell lung cancer.

Hereinafter, the present invention will be described in more detail.

Hereinafter, the present invention will be described in more detail.

The present invention provides a marker composition for diagnosing and predicting prognosis of small cell lung cancer comprising a single nucleotide polymorphism (SNP) of rs7559435 located at the 501st nucleotide sequence of the mitochondrial fission factor (MFF) gene represented by the nucleotide sequence of SEQ ID NO: 1. to provide.

While the present inventors were researching to discover biomarkers that can rapidly and accurately diagnose and predict the prognosis of small cell lung cancer, the difference in expression in small cell lung cancer was investigated through the search for EpiRequlome (Epigenetic regulatory elements) of SCLC and the establishment of an integrated database. I selected 296 polymorphic biomarkers (Epigenome regulatory SNPs) present in the gene search and histone modification binding region and correlated the selected single nucleotide polymorphism with the clinical results of 261 small cell lung cancer patients who received chemotherapy. As a result of analyzing whether the MFF gene rs7559435 polymorphism is significantly related to the prognosis of small cell lung cancer, the present invention was completed.

As used herein, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition. Among them, the present invention particularly includes predicting recurrence, progression, metastasis, and the like of small cell lung cancer. Therefore, an index that can accurately predict the recurrence and progression of small cell lung cancer is very important, and a factor that can predict the response to treatment while supplementing clinical indicators such as the degree of differentiation and stage of tissue is needed. Since it functions as an indicator, it can be used as a diagnostic factor for small cell lung cancer. That is, measurement of polymorphism of these genes can be used as a useful indicator (diagnostic marker) for predicting the degree of differentiation, stage, and progression of small cell lung cancer.

As used herein, the term "diagnosis marker or diagnostic marker" refers to a substance that can differentiate and diagnose cells with small cell lung cancer from normal cells, and shows an increase in cells with small cell lung cancer compared to normal cells. organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

As used herein, the term “EpiRequlome (Epigenetic regulatory elements)” refers to an element that regulates an epigenome. For example, it exists in a region where no protein is coded or is located in a distant region, so it does not directly affect the expression of adjacent genes, but it changes allele-specifically DNA methylation, DNA folding pattern, DNase I activity, etc. It refers to a number of many variants that retain their characteristics and roles.

As used herein, the term “mitochondrial fission factor (MFF)” refers to an external mitochondrial membrane protein that binds to GTPase Drp1, and is known to control the division of mitochondria and control the shape of peroxisome.

In addition, rs7559435 may be a single nucleotide polymorphism (SNP) present in the promoter region of the MFF gene.

Subsequently, the present invention provides a composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy comprising an agent capable of detecting a single nucleotide polymorphism (SNP) located in the 501st nucleotide sequence of SEQ ID NO: 1 do.

In addition, the agent is a primer or probe capable of amplifying a polynucleotide consisting of 10 to 100 consecutive bases comprising a single nucleotide polymorphism (SNP) located in the 501st nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide thereof can be

In addition, the single nucleotide polymorphism (SNP) located at the 501st nucleotide sequence of SEQ ID NO: 1 refers to the above-described rs7559435, and can be used as a marker for predicting survival prognosis of small cell lung cancer patients.

As used herein, the term “prognosis” refers to the progress and cure of neoplastic diseases such as lung cancer, such as the onset, recurrence, metastatic spread, and the possibility of lung cancer-induced death or progression, including drug resistance. do. For the purpose of the present invention, the prognosis refers to the risk of developing lung cancer, preferably small cell lung cancer, and the survival prognosis after the onset, preferably a patient treated for lung cancer with chemotherapy, more preferably small cell lung cancer with chemotherapy. It refers to the prognosis of the treated patient.

As used herein, the term "prediction" refers to determining whether a patient is likely to develop lung cancer, and reacting favorably or unfavorably to a treatment such as chemotherapy or radiation therapy to treat the patient, for example, a specific therapeutic agent; and/or the survival and/or likelihood of survival after surgical removal of the primary tumor, and/or treatment with chemotherapy for a specified period of time without cancer recurrence.

As used herein, the term "genetic polymorphism" refers to a case in which a genetic mutation appears at a frequency of at least 1% or more in a population. The insertion, deletion, or substitution of one nucleotide in DNA is called single nucleotide polymorphism (SNP).

As used herein, the term "polymorphism" refers to an arrangement in the sequence of a gene that varies within a population. Polymorphisms consist of different “alleles”. The placement of such polymorphisms can be identified by their position in the gene and the different amino acids or bases found therein. These amino acid variations are the result of two different alleles, two possible variant bases, C and T. Because the genotype consists of two different distinct alleles, any of several possible variants can be observed in any one individual (eg, CC, CT or TT in this example). Individual polymorphisms are also known to those skilled in the art and are used, for example, in the Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation available on the NCBI website. Identifier ("reference SNP", "refSNP" or "rs#").

As used herein, the term “allele” or “allele” refers to several types of one gene present at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles (bialele).

The term “rs_id” used in the present invention refers to rs-ID, an independent marker, assigned to all SNPs initially registered by the NCBI, which has been accumulating SNP information since 1998. In the present invention, it was described in the same form as rs831571. rs_id described in this table means a SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the position and sequence of the SNP using the rs_id. The specific sequence corresponding to rs_id, which is NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number, may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to such altered sequences.

As used herein, the term "single nucleotide polymorphism (SNP)" refers to a case in which a single nucleotide (A, T, C or G) in the genome differs between members of a species or between pairs of chromosomes of an individual. refers to the diversity of DNA sequences occurring in For example, when three DNA fragments from different individuals (eg, AAGT[A/A]AG, AAGT[A/G]AG, AAGT[G/G]AG) contain differences in a single base, Called two alleles (C or T), in general almost all SNPs have two alleles. Within a population, SNPs can be assigned to a minor allele frequency (MAF; the lowest allele frequency at a locus found in a particular population). A single base may be changed (replaced), removed (deleted) or added (inserted) into the polynucleotide sequence. SNPs can cause changes in the translation frame.

The term "marker for predicting survival prognosis of small cell lung cancer patients" as used in the present invention refers to a marker having polymorphisms capable of predicting the risk of small cell lung cancer, whether or not the onset of small cell lung cancer is cured, or the course, preferably in the above means the nucleotides described. In addition, the patient refers to a patient for determining the risk of developing small cell lung cancer or a patient who has undergone chemotherapy for small cell lung cancer.

As used herein, the term "subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial that do not show any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.

As used herein, the term “tissue or cell sample” refers to a collection of similar cells obtained from the tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; blood or any blood component; The cells may be at any time of conception or development in a subject. A tissue sample may also be a primary or cultured cell or cell line.

As used herein, the term "nucleic acid" is meant to include any DNA or RNA, for example, chromosomal, mitochondrial, viral and/or bacterial nucleic acids present in a tissue sample. It includes one or both strands of a double-stranded nucleic acid molecule, and includes any fragment or portion of an intact nucleic acid molecule.

As used herein, the term "gene" refers to any nucleic acid sequence or a portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. A gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to the gene.

As used herein, the term "primer" is a starting point for the stepwise synthesis of polynucleotides from mononucleotides by the action of a nucleotidyl transferase that hybridizes to a complementary RNA or DNA target polynucleotide and occurs, for example, in a polymerase chain reaction. It refers to an oligonucleotide sequence that functions as

As used herein, the term "protein" also includes fragments, analogs and derivatives of proteins that retain essentially the same biological activity or function as the reference protein.

As used herein, the term “treatment” refers to an approach for obtaining beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (ie, not worsening) of the disease state, delay or reduction in the rate of disease progression, disease state improvement or temporary alleviation and alleviation (partial or total), detectable or undetectable. "Treatment" may also mean increasing survival as compared to the expected survival rate if not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or prophylactic methods. Such treatments include the treatment required for the disorder being prevented as well as the disorder that has already occurred. "Palliating" a disease means that the extent and/or undesirable clinical signs of the disease state are reduced and/or the time course of progression is delayed or prolonged, compared to no treatment. means to lose

Furthermore, the present invention provides a kit or microarray for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy, comprising the composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy.

The kit of the present invention can be used to predict the survival prognosis of small cell lung cancer by identifying the rs7559435 polymorphic region (SNP) of the MFF gene, which is a marker for predicting the survival prognosis of small cell lung cancer. In the kit for predicting the survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy of the present invention, there is a polynucleotide, primer or probe for identifying the rs7559435 polymorphic region (SNP) of the MFF gene, as well as one or more suitable analysis methods. Any of these other component compositions, solutions, or devices may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the SNP, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. enzymes such as DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

In addition, the microarray of the present invention may be composed of a conventional microarray except for including the polynucleotide, primer or probe of the present invention.

Hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is, for example, labeling the nucleic acid sample with a labeling material capable of generating a detectable signal including a fluorescent material, for example, a material such as Cy3 and Cy5, and then hybridizing on a microarray and the labeling material. A hybridization result can be detected by detecting a signal generated from

In addition, the microarray method can simultaneously study the RNA expression of thousands or even tens of thousands of genes within a tumor, allowing more effective insight into the molecular basis of human disease.

It can also be used to evaluate gene expression patterns in tumor classification, clinical outcomes, and response to chemotherapy.

In addition, the present invention predicts the survival prognosis of small cell lung cancer patients or the prediction of treatment response to chemotherapy, including the step of confirming the rs7559435 polymorphism present in the promoter region of the mitochondrial fission factor (MFF) gene with respect to the nucleic acid extracted from the sample. It provides a way to provide information for

In addition, the method may further include determining that when the genotype of the rs7559435 polymorphism of the MFF gene is GA or AA, the chemotherapy response rate is higher and the survival prognosis is better than when the genotype is GG.

As used herein, the term “genotype” refers to a specific allele of a specific gene in a cell or tissue sample.

As used herein, the term "allele" or "allele" refers to one of two or more alternative forms of a gene occupying the same chromosomal locus.

In addition, in the method of providing information for predicting the survival prognosis of the small cell lung cancer patient or the treatment response to chemotherapy according to the present invention, the method for confirming the rs7559435 polymorphism of the MFF gene is a method of confirming the genotype of the rs7559435 polymorphism The genotype can be confirmed by sequencing analysis, sequencing analysis using an automatic sequencing analyzer, pyrosequencing, hybridization by microarray, PCR-RELP method (restriction fragment length polymorphism), PCR-SSCP method (single strand conformation polymorphism), PCR-SSO method (specific sequence oligonucleotide), ASO (allele specific oligonucleotide) hybridization method combining PCR-SSO method and dot hybridization method, TaqMan-PCR method, MALDI-TOF/MS method, RCA Method (rolling circle amplification), HRM (high resolution melting) method, primer extension method, Southern blot hybridization method, it can be carried out by a known method such as a dot hybridization method.

In addition, the results of the SNP polymorphism can be statistically processed using statistical analysis methods commonly used in the art, for example, Student's t-test, chi-square test (Chi-) square test), linear regression line analysis, and multiple logistic regression analysis, such as continuous variables, categorical variables, odds ratio, and 95 It can be analyzed using variables such as % confidence interval.

In addition, the method relates to examining the expression characteristics of specific markers according to small cell lung cancer, and the method disclosed herein is convenient for obtaining useful data and information when evaluating an appropriate or effective therapy for treating a patient with small cell lung cancer, It would be possible to provide an efficient and cost effective means.

In addition, the nucleic acid of a patient with small cell lung cancer can be obtained from samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine obtained from these patients, and the nucleic acid sample is obtained from DNA, mRNA, or mRNA. Contains cDNA that is synthesized.

In addition, the nucleic acid of the small cell lung cancer patient can be performed by conventional methods and separation methods such as phenol/chloroform extraction and protease K treatment, and can also be obtained by amplifying the target nucleic acid through PCR and purifying it.

In addition, the present invention can also provide information for determining the amount and administration method of an anticancer agent required to prevent recurrence or metastasis after chemotherapy treatment for small cell lung cancer.

That is, the present invention confirms the rs7559435 polymorphism of the MFF gene from a biological sample isolated from a patient with small cell lung cancer, and provides a method for administering an anticancer agent, such as the type, amount and concentration of the anticancer agent to be additionally administered depending on the case where the prognosis is poor or not, the corresponding lung cancer It can be applied differently from each other so as to be suitable depending on the type of

As such, the present invention includes all uses of the rs7559435 polymorphic expression profile of the MFF gene in small cell lung cancer patients as a prognostic and predictive marker for small cell lung cancer.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to embody the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Experimental method

1-1. Selection of research subjects

This study enrolled 261 patients who underwent chemotherapy after being diagnosed with SCLC at Kyungpook National University Hospital from 1997 to 2017. To prevent the effects of radiation therapy on chemotherapy, patients who received both chemotherapy and radiation therapy as a primary treatment method were excluded. Samples and clinical information used in this study were provided in accordance with the National Biobank Institutional Review Board (IRB) approved protocol of Kyungpook National University Hospital, and were performed with the consent of all patients. Patients treated with etoposide and cisplatin or irinotecan and cisplatin as first-line chemotherapy were the subjects of this study. For etoposide, 100 mg/m ² was administered intravenously to the patient on days 1 to 3 every 3 weeks, and cisplatin 60 mg/m ² was administered on the first day. Irinotecan was administered at a dose of 60 mg/m ² on days 1, 8, and 15 every 4 weeks, and cisplatin at a dose of 60 mg/m ² was administered intravenously on the first day. The decision to maintain treatment was based on the patient's disease progression, major toxicity, or the patient's or physician's decision. Assessment of tumor response was performed by computed tomography scans every 2 cycles. Response was assessed using response evaluation criteria in solid tumors (Eisenhauer EA, Therasse P, Bogaerts J et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228 -247.). The best overall response was reported for each patient and all responses were reviewed by a separate radiologist. Patients with complete response (CR) and partial response (PR) to first-line chemotherapy were considered responders, and patients with stable disease (SD) and progressive disease (PD) were considered non-responders.

1-2. EpiRegulome search and integrated DB construction

ChIP-seq was performed to confirm the histone modification status of small cell lung cancer. The results are shown in FIG. 1 .

As shown in FIG. 1 , in the small cell cancer cell line H146, H3K4me3 protein bound to the entire 17998 region, 85% was found in the promoter, H3K27Ac protein bound to the entire 19692 region, and 29% was found in the promoter.

Then, eQTL data using TCGA data was acquired through the PancanQTL database.

We obtained a list of 204,146 polymorphisms with cis-acting eQTL information in squamous cell lung cancer tissue and 259474 polymorphisms with cis-acting eQTL information in lung adenocarcinoma tissue.

Histone modification status data were obtained using the Public DB (ENCODE, Roadmap project), and subsequent analysis was performed using this data.

1-3. Selection of candidate biomarkers

The data profiling results were integrated and analyzed to select candidate biomarkers for small cell lung cancer.

First, by integrating the ChIP-seq data and eQTL results obtained in Example 1-2, 929 candidate polymorphic biomarkers (Epigenome regulatory SNPs) that exist in the histone modification binding region and have eQTL data in lung cancer tissues were selected. Subsequently, 749 candidate biomarkers were selected by using HapMap and NCBI SNP database, excluding SNPs having an East asia frequency of 10% or less among the 929 candidates, and then the SNPinfo site (https://snpinfo.niehs.nih.gov) By selecting TagSNPs using the LD Tag SNP selection menu, 296 candidate biomarkers were selected excluding LD SNPs (see FIG. 2).

1-4. Statistical processing

All these statistical procedures were performed using statistical software (SAS, Ver. 9.4, SAS institute, Cary, NC, USA). The Hardy-Weinberg equilibrium was tested using the goodness-of-fit χ2 test. Differences in genotype distribution according to patient characteristics were compared using the chi-square test. For survival assessment, the time between the date of first chemotherapy and the date of death was measured as overall survival (OS). Estimated survival according to different genotypes and clinical variables was analyzed using the Kaplan-Meier method and log-rank test. Hazard ratio (HR) and 95% confidence intervals (CIs) were calculated using multivariate Cox proportional hazards models. Treatment response analysis to chemotherapy was calculated using the odds ratio (OR) and 95% confidence intervals (CIs) through a logistic regression test. A cut-off P=0.05 was adopted for all statistical analyses, and all clinical factors were adjusted as follows; Gender (male vs female), smoking status (none smoker / smoker / current smoker), clinical stage (limited disease versus widespread disease), ECOG PS (0-1 versus 2), weight loss (yes versus no), secondary- line chemotherapy (yes vs. no), whether or not to treat the tumor with radiation (yes vs. no).

<Example 2> Biomarker discovery

2-1. Genotyping Results

Samples of 261 patients with small cell lung cancer were genotyped using Sequenom MassARRAY. The results are shown in Table 1.

As shown in Table 1, in both cohort groups, ECOG (eastern cooperative oncology group) and anticancer drug administration (regimen) were found to be associated with treatment response to chemotherapy, and age, stage of lung cancer ( stage), eastern cooperative oncology group (ECOG), chemotherapy, and radiation to tumor were found to be related to overall survival.

2-2. Discovery of biomarkers predicting chemotherapy response in small cell lung cancer patients

The correlation between the 296 candidate biomarkers selected in Example 1 and the response to chemotherapy in small cell lung cancer patients was analyzed. As a result of comparative analysis of the dominant model with major allele homozygote and heterozygote, the result of comparative analysis of the recessive model with minor allele homozygote and heterozygote, the codominant model showed a codominance effect. The results are shown in Table 2.

For example, in the case of rs7559435, there were 141, 96, and 22 patients with the genotypes of major allele homozygote, heterozygote, and minor allele homozygote, respectively, and treatment with chemotherapy in the Dominant Model (2+3vs1) It was confirmed that there was a significant association with the response (OR=2.56, 95% CI=1.36-4.84, p=0.0038). These results mean that when the rs7559435 genotype of small cell lung cancer patients has GA or AA, the treatment response rate to chemotherapy is 2.56 times higher than that of the GG genotype.

In addition, 24 SNPs highly correlated with chemotherapy response among 296 SNPs were discovered (Table 2).

Shades of red: p<0.05

1: major allele homozygote

2: heterozygote

3: minor allele homozygote

2-3. Discovering biomarkers that predict the prognosis of small cell lung cancer patients

The correlation between the 296 candidate biomarkers selected in Example 1 and the survival rate of small cell lung cancer patients was analyzed. The results are shown in Table 3.

For example, in the case of rs7559435, there were 141, 96, and 22 patients with the genotypes of major allele homozygote, heterozygote, and minor allele homozygote, respectively, and there was a significant correlation with survival rate in the Dominant Model (2+3vs1). (HR=0.73, 95% CI=0.55-0.98, p=0.0382). These results mean that the survival rate of small cell lung cancer patients with rs7559435 genotype GA or AA is higher than that of GG genotype.

In addition, among 296 SNPs, 47 SNPs highly correlated with chemotherapy response were discovered.

Shades of red: p<0.05

1: major allele homozygote

2: heterozygote

3: minor allele homozygote

2-4. Comprehensive analysis results

Through Example 2-2, 25 SNPs highly correlated with the treatment response to chemotherapy for small cell lung cancer were discovered, and 48 SNPs highly correlated with the survival rate of small cell lung cancer were discovered through Example 2-3. Table 4 shows the results of comprehensive analysis of the results of Examples 2-2 and 2-3, and has a p value of 0.05 or less in the same model in the chemotherapy response rate and survival rate, which affects the survival rate according to the chemotherapy response Only SNPs judged to have an impact on

As shown in Table 4, among them, rs7559435, a SNP present in the promoter of the MFF protein, showed p values of 0.0038 and 0.0382, respectively, in the Dominant Model of the chemotherapy response rate and survival rate of small cell lung cancer. It was confirmed that there was a close correlation with the survival rate. These results show that rs7559435, a SNP present in the promoter of the MFF protein, is closely related to the response and survival rate after chemotherapy of small cell lung cancer depending on the presence or absence of the allele. This means that it can be used as a biomarker for predicting the prognosis of therapy.

As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the claims described below rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Diagnostic methods for prognosis of small-cell lung cancer using MFF SNPs <130> 2020-0485-KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1001 <212> DNA <213> Homo sapiens <220> <221> variation <222> (501) <223> y is G or A <400> 1 agttcttaaa tttatttgat tgatgtctca tgcctcccta aaatacataa aaccaagctg 60 cgccccaacc accttggaca catgttctca ggacctcctg agggctctgt caggggccat 120 ggtcactcac atttggctca gaataaatct cttcaaatat tttagagttt gactcttttc 180 ctcatcagtt taattcttag gcccagccaa gaccccaaga ggggagagga aatattttca 240 ctttccctac accagtgtta agaagcccaa gaacgcaatt cagagggctt cccaaatcat 300 tttgaagaat ggtaacatct ctagaatatc aattagatcg taggggagta cataagtcat 360 gtgtacacct ttaggtgttt gtgaaaacca aggcatataa aatatcacat tatttggcct 420 cagtacattc ccatactgac aagcactgta ccatttttcc cccgctctaa gcaagggctt 480 cctgggggga agagtctgtg yttgggtgtt cagaagcaga gagtgctctc ccaaagctaa 540 acgatgccgc caactactgt gaaacttaac ttcccctctc ccggaattct catggaagac 600 aaggtctgat caaccgaggg cttaagaagt atcaaattgg aactcatgga atcactggcg 660 gagctgattt acatatgaga attcctaagc ctcacagcat agaaattttc atgccagatg 720 gtcctgaaaa agttgtttta attggctacc aaaggtttac cttgcaaaca ccaagttctt 780 aaagattaca aaatctaata aagccgaagt gtgaattaaa cttaaccgca agaggagtca 840 cctgcggtca cctcgtttcg cccacaacaa agatgtctgc gcatcgctta ccgtggagcc 900 gttccagtgc gtgggccacg cgtccgccct tttccgcgtc acatgaccac gaacactctt 960 ccgctacggc tcccagaagg ggccagcccg cgcctttcgc g 1001

Claims (8)

A composition for markers for diagnosing and predicting prognosis of small cell lung cancer, comprising a single nucleotide polymorphism (SNP) of rs7559435 located at the 501st nucleotide sequence of the mitochondrial fission factor (MFF) gene represented by the nucleotide sequence of SEQ ID NO: 1.
The method of claim 1,
The single nucleotide polymorphism (SNP) of rs7559435 is a marker composition for diagnosis and prediction of prognosis of small cell lung cancer, characterized in that it exists in the promoter region of the MFF gene.
A composition for predicting survival prognosis of small cell lung cancer patients or therapeutic response to chemotherapy, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) located in the 501st nucleotide sequence of SEQ ID NO: 1.
4. The method of claim 3,
The agent is a primer or probe capable of amplifying a polynucleotide consisting of 10 to 100 consecutive bases containing a single nucleotide polymorphism (SNP) located in the 501st nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide thereof. A composition for predicting the survival prognosis of small cell lung cancer patients or treatment response to chemotherapy, characterized in that.
A kit for predicting survival prognosis of small cell lung cancer patients or treatment response to chemotherapy, comprising the composition according to claim 3 or 4.
A microarray for predicting the survival prognosis of a patient with small cell lung cancer or a therapeutic response to chemotherapy, comprising the composition according to claim 3 or 4.
With respect to the nucleic acid extracted from the sample, the survival prognosis of small cell lung cancer patients, including the step of confirming the rs7559435 polymorphism present in the promoter region of the mitochondrial fission factor (MFF) gene, or information for predicting treatment response to chemotherapy. How to.
8. The method of claim 7,
When the genotype of the rs7559435 polymorphism of the MFF gene is GA or AA, the survival prognosis of a small cell lung cancer patient, characterized in that it further comprises the step of determining that the chemotherapy or survival prognosis is better than when the genotype is GG or a method of providing information for predicting a therapeutic response to chemotherapy.

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