TWI607091B - Method or kit for determining lung cancer development - Google Patents

Description

Method and kit for detecting lung cancer

The present disclosure relates to the field of cancer development. More specifically, the present disclosure relates to a method or kit for assessing lung cancer risk in a subject.

The similarity between deoxyribonucleic acid (DNA) and individual is about 99.9%. The individual's unique phenotype is determined by the remaining 0.1% difference. In addition to causing phenotypic differences, the above-mentioned "small" differences are also related to whether an individual will develop a disease. There have been many studies wishing to understand the association between the risk of certain diseases and the genetic or single nucleotide polymorphism (SNP) of a particular variant. A SNP refers to a base position in which a single base substitution occurs at least 1% in a population; in a human genome, an SNP occurs on average about 1,900 base pairs.

Several SNPs are known to be involved in cancer development. For example, it is located in the epidermal growth factor (epidermal growth factor, EGF) gene s4444903 SNP may lead to gallbladder cancer and lung cancer; breast cancer 1 (BRCA1) gene and BRCA2 gene rs1799950 and rs1799954 SNP are associated with breast cancer; bone marrow peroxidase (myeloperoxidase) , MPO) gene rs2333227 SNP may cause gastric cancer and lung cancer; and the SNP of rs3218536 located in the X-ray repair cross complementing 2 (XRCC2) gene may play a role in the occurrence of breast cancer and ovarian cancer An important role.

Yes-associated protein 1 (YAP1), also known as YAP or YAP165, is encoded by the YAP1 gene located on human chromosome 11q22. YAP1 is a multifunctional intracellular junctional protein and transcriptional co-activator. The abnormal expression of YAP1 may cause abnormal intracellular signaling, which may lead to different diseases. Abnormal performance of YAP1 is known to be associated with multiple types of cancer. For example, excessive expression of YAP1 can be observed in different human cancers including liver cancer, esophageal squamous cell carcinoma, non-small cell carcinoma, and ovarian cancer. The inappropriate expression of YAP1 in the nucleus and cytoplasm is associated with the development of colon adenocarcinoma, lung adenocarcinoma and ovarian serous cystadenocarcinoma. In addition, even a single S127A mutation in YAP1 enhances the growth and metastasis of breast cancer and melanoma. Therefore, the abnormal performance of the YAP1 protein is often closely related to cancer.

Lung cancer is the most common cause of death in men and women. First, an average of 1-2 million people die each year from lung cancer. However, most methods for assessing the risk of lung cancer using genomic identification and/or protein expression are currently lacking in accuracy and effectiveness. Therefore, there is a need for a more accurate and effective method for assessing whether a subject has lung cancer or is at risk of developing lung cancer.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The present disclosure relates to a method for assessing whether a subject has lung cancer or is at risk of developing lung cancer; and a kit for assessing the risk.

One aspect of the present disclosure relates to a method for assessing whether a subject is suffering from lung cancer or is at risk of developing lung cancer from a biological specimen of a body. The method comprises extracting DNA from the biological sample; and detecting whether the extracted DNA has a single nucleotide polymorphism (SNP) locus of rs193100333; wherein, if the extracted DNA has rs193100333 The SNP locus represents the risk of lung cancer or lung cancer in the individual.

In accordance with certain embodiments of the present disclosure, the method further comprises amplifying the extracted DNA using a pair of primers having nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

According to certain embodiments of the present disclosure, the SNP locus can utilize direct sequencing, primer extension, dynamic allele-specific hybridization (DASH), molecular letter drift (molecular beacon), SNP microarray, restriction fragment length polymorphism (RFLP), quantitative polymerase chain reaction (qPCR), petal endonuclease (flap) Endonuclease, FEN), single strand conformation polymorphism, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography (DHPLC), full A high-resolution melting of the entire amplicon or a DNA mismatch-binding protein is amplified for detection. In an embodiment of the present disclosure, a primer extension method is used to detect a SNP locus, wherein the primer assisted laser desorption ionization time-of-flight mass is used. Spectrometry, MALDI-TOF MS) for detection.

According to some embodiments of the present disclosure, the biopsy may be a skin slice, a whole blood sample, a skin color layer (buffy) Coat) specimen, plasma specimen, serum specimen, urine specimen or mucus specimen.

According to an embodiment of the present disclosure, the individual is a Chinese.

In accordance with certain embodiments of the present disclosure, the SNP locus of rs193100333 corresponds to a mutation in a YAP1 protein in which arginine at protein position 331 is substituted with tryptophan.

Another aspect of the present disclosure relates to a kit for detecting a SNP locus of rs193100333 in a biological specimen derived from a body to assess whether the individual is at risk of developing lung cancer or having lung cancer. The kit comprises a pair of primers having nucleotide sequences of SEQ ID NO: 1 and 2, respectively, which can be amplified by a polymerase chain reaction (PCR) to amplify a SNP locus comprising rs193100333 in a biological sample. a DNA fragment; and a PCR reaction reagent comprising PCR reaction enzyme, MgCl ₂ , deoxynucleotide triphosphate (dNTP), PCR reaction buffer, and double-distilled water.

In certain embodiments of the present disclosure, the kit further comprises a DNA template as a positive control, the nucleotide sequence of the DNA template having a SNP locus of rs193100333. In other embodiments of the present disclosure, the kit further comprises a DNA template as a negative control, the nucleotide sequence of the DNA template having no SNP locus of rs193100333.

The technology of the present invention after referring to the following embodiments The basic spirit and other objects of the invention, as well as the technical means and implementations of the invention, are readily apparent to those skilled in the art.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; ) is the genealogy of the original family, and (B) is the genealogy of the validation cohort; the arrow is labeled as the proband, the gender is represented by the shape symbol, the square represents the male, and the circle represents the Female; +, x, and NA in the shape symbol represent YAP1 R331W carrier, non-container, and DNA unreachable; the shape symbol marked by a diagonal line indicates that the individual has died; The numbers represent the age of the individual; and Figure 2 is a histogram depicted in accordance with an embodiment of the present disclosure, wherein (A) is the colony formation ability of a particular cell, and (B) is specific Cell invasion ability.

The various features and elements in the figures are not drawn to scale, and are in the

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method. Ranges of values are expressed herein as being from one endpoint to another or between two endpoints; unless otherwise stated, the numerical ranges recited herein are inclusive.

Unless otherwise defined in this specification, the science used here The meanings of the technical terms are the same as those of ordinary skill in the art to which the present invention pertains. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

In the present specification, "single nucleotide polymorphism" (SNP) refers to a DNA sequence variation; more specifically, the DNA sequence variation refers to a different individual in a species, or Between a pair of chromosomes, a single nucleotide (A, T, C, or G) in a genomic (or other shared sequence) will vary from individual to chromosome. In general, the Single Nucleotide Polymorphism Database is affiliated with the National Center for Biotechnology Information (NCBI) and the National Human Genome Research Institute (NHGRI). , db SNP) assigns a SNP reference identifier (rs) to each SNP.

"Nucleotide sequence", "polynucleotide" and "nucleic acid" are interchangeable words in this specification, and in accordance with the disclosure, A double strand of DNA, a single strand of DNA, or a transcription product of the DNA (eg, a ribonucleic acid (RNA) molecule). The nucleic acid, polynucleotide or nucleotide sequence of the present invention can be extracted and purified by an isolation method or a genetic engineering method (or Partially purified); the separation method includes, but is not limited to, ion-exchange chromatography and molecular size exclusion chromatography; the genetic engineering method may be amplification. Subtractive hybridization, cloning, sub-cloning, chemical synthesis or a combination of these genetic engineering methods.

The term "subject" as used in this specification refers to mammals (including humans) that benefit from the methods of the present disclosure. In addition, the term "subject" includes both male and female, unless otherwise indicated. According to an embodiment of the present disclosure, the individual is a Chinese.

The present disclosure relates to a method for assessing risk by assessing the risk of lung cancer in a body by detecting the SNP locus of rs193100333; and a kit for detecting the SNP locus of rs193100333.

One aspect of the present disclosure relates to a method of assessing whether a subject is suffering from lung cancer or is at risk of developing lung cancer from a biological specimen derived from a body. The method comprises extracting DNA from the biological sample; and detecting whether the extracted DNA has a SNP locus of rs193100333; wherein if the extracted DNA has a SNP locus of rs193100333, it indicates that the individual has lung cancer or has lung cancer risk. According to some embodiments of the present disclosure, the method further comprises utilizing a pair of specific sequence numbers: nucleotide sequences of 1 and 2, respectively The primers of the column are used to amplify the extracted DNA. In the PCR reaction, the pair of primers can specifically amplify a DNA fragment containing the SNP locus of rs193100333. The amplified DNA fragment can be used to detect the SNP locus of rs193100333 and increase the accuracy of the risk assessment of the method of the invention.

In accordance with an embodiment of the present disclosure, a test can be utilized to detect the SNP locus of rs193100333, which is selected from direct sequencing, primer extension, dynamic dual-specific hybridization (dynamic Allele-specific hybridization (DASH), molecular beacon, SNP microarray, restriction fragment length polymorphism (RFLP), quantitative polymerase chain reaction (quantitative polymerase chain reaction, qPCR), flap endonuclease (FEN), single strand conformation polymorphism, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography A group consisting of a high-resolution melting of the entire amplicon or a DNA mismatch-binding protein.

In one embodiment of the present disclosure, the whole genome of an individual is directly sequenced to detect the SNP locus of rs193100333. Whole-genome sequencing can be performed using the next generation sequencing assay (NGS assay). NGS first cleaves a single genomic DNA to form a library containing different small fragments, and then analyzes the sequences of the small fragments uniformly and accurately with millions of parallel reactions. A confirmed base string (called a sequence read, read) can be recombined (reordered) using a known reference gene (as a scaffold) or in a non-referenced genome In the case of direct reorganization (re novo sequencing). The recombined aligned sequence reads can be used to read the full length sequence of each chromosome in the genomic DNA.

In another embodiment of the present disclosure, the primer extension method is used to detect the SNP locus of rs193100333. The primer assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used for the primer extension method. Mass spectrometry is a technique for sensitively distinguishing the molecular weight of each component in a heterogeneous aggregate of biomolecules, and further confirming each component. The MALTI-TOF MS is to place the sample to be analyzed in a UV-absorbing matrix pad, and then irradiate the sample with a short laser pulse. The ionized molecules accelerate away (ie, desorb) the matrix spacer and move to an electric field toward the detector. The "time of flight" to the detector is determined by the weight/charge (m/z) ratio of each molecule. When DNA sequencing is performed using MALTI-TOF MS, the DNA sequence to be sequenced is first transcribed into RNA in vitro by four independent reactions (each reaction containing three rNTP bases and one specific dNTP). . RNA hydrolases are unable to cleave the location of dNTP insertion in the transcribed RNA, thus producing different fragments. Each fragment has a specific m/z ratio that forms a specific peak on the MALTI-TOF spectrum. The MALTI-TOF quality signal measured by the DNA sample is then compared to the expected m/z spectrum of the reference sequence (containing the products of the four cleavage reactions). Differences in SNP between any of the sample DNA and the reference DNA sequence will produce a predictable translation in the spectrum to infer its exact properties.

In yet another embodiment of the present disclosure, a quantitative polymerase chain reaction (qPCR) is utilized to detect a particular SNP locus. qPCR uses a combination of primers that can be labeled separately to each of the dual genes to amplify and simultaneously detect and quantify DNA samples containing the SNP locus. Compared to the amplification period of other SNPs, a well-designed primer can amplify its target SNP at an earlier cycle. Each SNP is detected by an individual qPCR reaction to distinguish two or more dual genes. To achieve a sufficiently high degree of specificity, an artificial mismatch can be placed near the 3' end of the primer sequence, a technique commonly referred to as Taq-MAMA. Compared to a single mismatch, the artificial mismatch causes a larger amplification delay for the non-targeted dual gene and does not substantially affect the amplification reaction of the target SNP.

In still another embodiment of the present disclosure, the package is A specific DNA fragment containing the SNP locus of rs193100333 was directly sequenced to detect the SNP locus.

According to an embodiment of the present disclosure, the biological specimen may be a skin slice, a whole blood sample, a buffy coat sample, a plasma sample, a serum sample, a urine sample, or a mucus sample. Preferably, the whole blood sample is used as a biological sample; more preferably, the white blood cells and platelets obtained from the whole blood sample are used as biological samples.

In accordance with an embodiment of the present disclosure, the SNP locus of rs193100333 corresponds to a mutation in a YAP1 protein in which the arginine at position 331 of the protein is substituted for tryptophan.

Another aspect of the present disclosure relates to a kit for detecting a SNP locus of rs193100333 in a biological specimen of a body, thereby assessing whether the individual is at risk of developing lung cancer or having lung cancer. The kit includes a pair of primers, a PCR reaction reagent, and instructions for indicating how the user uses the kit. The pair of primers respectively have the nucleotide sequences of SEQ ID NO: 1 and 2, and the DNA fragment containing the SNP locus of rs193100333 in the biological sample can be amplified in a PCR reaction. The PCR reaction reagent comprises PCR reaction enzyme, MgCl ₂ , deoxynucleotide triphosphate (dNTP), PCR reaction buffer and double distilled water.

According to some embodiments of the present disclosure, the kit further comprises a DNA template as a positive control, the nucleotide sequence of the DNA template having a SNP locus of rs193100333. According to other embodiments of the present disclosure, the kit further includes a negative control group. A DNA template in which the nucleotide sequence of the DNA template does not have a SNP locus of rs193100333.

The present disclosure relates to the association of the SNP locus of rs193100333 with the development of lung cancer. A number of experimental examples are presented below to illustrate methods for assessing whether a subject is suffering from or at risk of developing lung cancer; and a kit for conducting a risk assessment. The embodiments are only intended to be illustrative of the invention, and are not intended to limit the scope of the invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Materials and methods

Research group

The system involved in this study was selected from participants in the Genetic Epidemiology Study of Lung Adenocarcinoma (GELAC) and the Cancer Screening Project (CSP). The GELAC program includes lung cancer patients, family members of the disease, and matched healthy control individuals. Control individuals in GELAC were individuals randomly selected from the same hospital for health checkups during the recruitment period. In this study, all lung cancers were lung adenocarcinomas, and GELAC collected the genetic DNA of all patients and their families. CSP is a forward-looking research group of healthy individuals recruited from seven townships in Taiwan.

To confirm the SNP associated with lung cancer, by GELAC A family with six lung cancer patients was selected and their genomes were analyzed for whole genome sequencing. The mother and her four daughters in the family are diagnosed lung adenocarcinoma patients, and their sons are healthy individuals. The father also died of lung adenocarcinoma, but his DNA could not be obtained. None of the six lung cancer patients had a history of smoking.

After screening, 30 normal individuals selected from CSP were subjected to MALDI-TOF test analysis to remove SNPs that would be expressed in healthy individuals.

In addition, 1135 normal control individuals and 1312 lung cancer patients were analyzed to understand the association between SNP YAP1 R331W and lung cancer. All lung cancer patients are selected from the GELAC program. Of the 1,135 healthy individuals, 470 were selected from the CSP program and 665 were selected from the GELAC program.

The genomic DNA was extracted from the skin color blood layer of the peripheral blood of each specimen according to a standard treatment procedure. The study was conducted with the approval of the Institutional Review Board. Informed consent was also obtained for all patients in this study.

Gene sequencing by next generation sequencing assay (NGS assay)

Individual established SOLiD ^TM 4 system in accordance with the standard processing procedures (Life Technologies, Foster City, CA ) paired database (Mate-paired libraries). Briefly, the genomic DNA is first cleaved into fragments of about 3 kb in size to prepare a library that serves as a template for emulsion polymerase chain reaction (emulsion PCR). Using the adapter sequence (adapter sequence) linked to magnetic beads and the library fragments, followed system SOLiD ^TM EZ Bead ^TM (Life Technologies, Foster City, CA ) were transfected colonization amplification. The 3' end of the DNA fragment linked to the magnetic beads is modified to attach the magnetic beads to the slide. After the SOLiD ^TM sequencing primer hybridizing to the adapter sequence (hybridized), using four kinds of fluorescent labeled probes double bases of (di-base probe) connection type sequencing (ligation-based sequencing). Each nucleotide is sequenced in two different ligation reactions to increase the accuracy of the sequencing. The 50 base pairs of the forward tag (F3 tag) and the reverse tag (R3 tag) are respectively sequenced. Use according to instructions by SOLiD ^TM BioScope ^TM software to the reference sequence than the results of sequencing the human genome and (hg19).

Gene sequencing by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

According to the SNP gene sequencing method established by Su KY et al. (Prevention Epidermal Growth Factor Receptor (EGFR) T790M Mutation Predicts Shorter EGFR Tyrosine Kinase Inhibitor Response Duration in Patients With Non-Small-Cell Lung Cancer, Journal of Clinical Oncology, 30: 433-440, 2012), MALDI-TOF MS was performed using the MassARRAY system (SEQUENOM, San Diego, CA) to confirm the genomic variation detected by the NGS assay.

Gene sequencing by quantitative polymerase chain reaction (qPCR)

A TaqMan type qPCR assay (Life Technologies, Carlsbad, CA) was performed according to the instructions for use to genetically sequence the YAP1 R331W dual gene.

Gene sequencing of specific DNA fragments

Sequence analysis of specific DNA fragments and library construction was performed using Pacific Biosciences (Pacific Biosciences, Menlo Park, CA) according to the instructions for use. To construct a 5 kb insert database, the pair has a sequence number: 1 (when YAP1 rs193100333 is marked as +1, at -594 bp) and sequence number 2 (when YAP1 rs193100333 is marked as +1, at +4612 bp) The primer uses a PCR reaction to amplify 200 ng (ng) of genomic DNA. After purification and end-repair of the amplified DNA fragment, it was ligated to the SMRTbell sequencing adaptor. The sequenced library prepared was purified three times with 0.6 volumes of Agencourt AMPure XP magnetic beads (Beckman Coulter Genomics, Denver, CO). The SMRTbell sequencing library, sequencing primers, and polymerase were uniformly mixed, and Pacific Biosciences sequencing was performed using a PacBio RS instrument (sequence cycle of 120 minutes). The preliminary results were analyzed (Pacific Biosciences, Menlo Park, CA). All databases were sequenced using v.2 chemistry and v.1.3.3 analytical software (Pacific Biosciences, Menlo Park, CA) according to the operating instructions.

Biological data analysis

Sequence data including image analysis, comparison of human reference gene bodies (UCSC Hg19), and detection of single nucleotide mutations were analyzed using standard SOLiD software BioScope (Life Technologies, Foster City, CA). The genomic variation detected by the SIFT software annotation.

The invention comprises four steps for screening for risk alleles. The first step integrates the genetic relationship between family members of the six-cancerous lung cancer patient selected from the GELAC program described in the "Research Population" section. Since the father (who can't get his DNA), the mother and the four daughters are all lung adenocarcinoma patients, and the son is not a lung adenocarcinoma, it is inferred that at least one of the parents has a risky dual gene and inherits the gene. Give a daughter with lung adenocarcinoma and pass a non-risk gene to a non-sick son. Accordingly, the daughter may carry a risky dual gene of heterozygous or homozygous. Mutations detected by NGS and consistent with genetic characteristics were screened.

The second step was screened by MALDI-TOF MS analysis data. The MALDI-TOF MS platform was used to confirm the non-synonymous mutations obtained by the first screening step.

After eliminating the incorrect genotype identified by MALDI-TOF MS, the third step of screening compared the remaining candidate mutations to the published four genomes (including YH1, Korean, Watson, and Venter). Correct. Mutations of the four genomes were excluded.

In the final screening step, due to the assumption of pathogenic duality The gene has a very low dual gene frequency in Taiwan, so 30 normal subjects selected from CSP were genetically sequenced using MALDI-TOF MS to analyze candidate mutations. Mutations detected by any of the 30 normal individuals are further excluded to ensure a low incidence of disease candidate mutations in non-affected individuals. The findings of this study were confirmed in two steps using 1312 lung cancer patients and 1135 normal individuals. In the first confirmation step, 1135 normal individuals and 651 lung cancer patients were sequenced using MALDI-TOF MS to verify candidate mutations screened by the above four screening steps. In the second confirmation step, TaqMan-type qPCR sequencing was used to analyze the significant risk dual genes in the first step.

Cell colony formation assay

To understand the ability of the cells to grow non-attached, 0.7% agarose dissolved in physiological saline was placed on the bottom of the 6-well plate. Further, 0.3% agarose dissolved in a cell culture medium containing 10% fetal bovine serum was placed in the upper layer. A549 lung cancer cells were suspended in 1 ml of RPMI cell culture medium containing 0.35% low melting point agarose, and planted in the upper layer of the 6-well plate at a density of 2 x 10 ³ cells per well. Three weeks later, the wells were washed with physiological saline, fixed with 4% paraformaldehyde, and finally stained with 0.1% crystal violet stain. Thereafter, cell colonies exceeding 0.5 mm were counted in three independent experiments.

Basement membrane matrix invasion test (Matrigel Invasion Assay)

In this study, the Boyden cell was further used to evaluate the cell invasive ability; the Boden cell shaper consisted of two compartments filled with cell culture medium (the upper compartment and the lower compartment). A microporous polycarbonate membrane is interposed in the second compartment. A basement membrane matrix (Becton Dickinson, Franklin Lakes, NJ) was coated on a polycarbonate film (Costar, Cambridge, MA) with 8 micron pores and placed between the two compartments. 1 x 10 ⁴ A549 lung cancer cells were planted into the upper compartment of the shifter (this compartment contains 200 μl of serum-free cell culture), allowing it to move through the pores of the membrane to the lower compartment ( This compartment has a cell culture medium containing 10% fetal calf serum). After 16 hours of culture, the cells of the basement membrane-coated thin film were fixed with methanol, and stained with Giemsa solution (Sigma, St Louis, MO). The cells moved to the lower compartment were counted at a magnification of 200 times under an optical microscope. The experiment was repeated three times.

Statistical Analysis

Fisher's exact test was used to detect differences in frequency between different groups. Multivariate logistic regression was used to assess the risk of risky dual genes using individual age, gender, and smoking covariate. Statistical analysis was performed with SAS 8 (SAS Institute Inc., Cary, NC). Therefore, the tests are both two-tailed, and when the p value is <0.05, it means statistical difference.

Example 1 Confirmation of YAP1 R331W mutation in lung cancer patients

1.1 Gene sequencing and verification YAP1 R331W

To verify the association between SNP and lung cancer, a whole genome sequencing of a family of high-density lung adenocarcinomas (hereinafter referred to as "original family") selected from the aforementioned GELAC program, in which the family The mother (number: 502, indicated by the arrow) and her four daughters (number: 601-604) all suffered from lung cancer, while her son (number: 605) had no lung cancer (Fig. 1A). The average throughput of the NGS test was approximately 73 Gb with a 24 fold coverage. On average, approximately 3 million single nucleotide mutations were detected by the NGS assay for each body (Table 1).

In order to compare the differences between individuals with or without lung cancer, the dominant model was used for analysis. The results indicated that 70,827 SNPs were detected only in the mother and its four daughters, but not in the son's body. Measured. Among these SNPs, 240 non-synonymous dual genes can be identified.

Further, in the 30 normal Chinese individuals selected from the above CSP program, the 240 dual genes were analyzed by MALDI-TOF sequencing to exclude SNPs of normal individuals. After screening, there are 6 dual genes left, one of which is located in the exon 6 of the YAP1 gene of chromosome 11q. The SNP reference identification code for this SNP is rs193100333, which corresponds to a mutation in the YAP1 protein in which the arginine at position 331 of the protein is substituted for tryptophan.

Next, in order to verify the performance of YAP1 R331W in lung cancer patients, sequencing analysis was performed on 1312 lung cancer patients (hereinafter referred to as "validation cohort") and 1135 normal individuals by MALDI-TOF MS test and qPCR test. . As shown in Table 2, the low incidence of YAP1 R331W in normal individuals (0.18%) was consistent with the reported frequency of the Asian population of the 1,000 gene program (one person per 286 person, or 0.35%, the results are not shown). At the same time, in lung cancer patients, the incidence of mutations increased to 1.1% (p = 0.0095), and the odd ratio (OR) was 6.1. After adjusting for age, smoking, and gender variables, the OR was 5.9 (p=0.019) (Table 3). This ratio is much higher than the risk comparison value (<2.0) reported by the previous Genome Lung Cancer Institute.

These results indicate that the YAP1 R331W mutation can be used as a potential indicator for risk assessment of lung adenocarcinoma.

1.2 The genealogy of the lung adenocarcinoma family

1.2.1 Genealogy of the primary disease family

In view of the fact that genetic mutations and/or SNP loci are associated with the genetic susceptibility of diseases such as cancer, the YAP1 R331W performance of relatives of index cases in the primary disease family is further tracked, with arrows pointing to indicator cases (number: 502), and the family contains four generations (Fig. 1A). The YAP1 mutation was detected in all individuals who were able to obtain DNA. The father of the indicator case has two wives, so the indicator case has one sister and three half-brothers; two of them (numbers: 532 and 501) have been diagnosed with lung adenocarcinoma. Individual 532 carries the YAP1 mutation and the DNA sample of individual 501 is not available. However, it is speculated from the genetic model of YAP1 that the individual number: 501 should also be a mutational factor .

Individual number: 501 has five children. Of the four children who were able to obtain DNA, two were positive (numbers: 653 and 654) and then underwent low dose computed tomography (LDCT) scans. Individual number: 653 is a non-smoker, LDCT test results are positive, further examination found that suffering from stage I lung adenocarcinoma. As for the individual number: 654, diagnosed It was later found that the right upper lobe (RUL) of the lung had an 8 mm lesion of ground ground-glass opacity (GGO) and continued to be observed. The results of lung lesions in two non-contributors (numbers: 651 and 655) were negative.

Individual number: 532 (dead) There are three children who obtained their DNA for research and analysis (numbers: 656, 657, and 659). Individual number: 656 suffering from lung adenocarcinoma, but YAP1 wild-type carrier, the DNA obtained by its four children (number: 753, 754, 755 and 756) is negative for YAP1 mutation, and both For healthy individuals. Individual number: 657 with the YAP1 mutation, and its LDCT scan three years ago showed four 4 to 8 mm GGO lesions in the bilateral lung; however, the individual did not participate in subsequent interviews in this study. Individual number: 659 also has a YAP1 mutation, and the left lower lobe (LLL) of the lung has a 5 mm GGO lesion.

1.2.2 Verifying the genealogy of the ethnic group

Of the 1312 individuals in the validation cohort , 14 lung adenocarcinoma patients had a YAP1 mutation. Six of the family information can be obtained (Figure 1B). The genomic DNA of relatives of six patients was collected for YAP1 mutation detection.

In Family A, the brother with the YAP1 mutation (number: 54) in the indicator case (number: 53, arrow marker) was diagnosed with advanced lung adenocarcinoma two months before the visit.

In Family E, LDCT scans revealed that the three YAP1 mutations (numbers: 61, 38, and 39) had GGO lesions in the lungs (the lower left lobe of the sisters had an 8 mm GGO lesion; The daughter had a 6 mm GGO lesion in the right upper lobe of the lung and a 5 mm GGO lesion in the left lower lobe of the lung. In contrast, the daughter with the wild-type gene (number: 40) had no GGO lesions.

In Family D, the three siblings of the indicator case (number: 57, arrow signer) had other types of cancer, but the researchers were unable to obtain their DNA samples. It is worth noting. In families D and E, the cases with the YAP1 mutant gene were breast cancer.

Based on the above findings, the primary disease family and the six new belt families can be classified into lung adenocarcinoma, GGO, cancer-free, and no-update health status according to the health status of their members. These results indicate that individuals with the YAP1 R331W mutant gene are significantly more likely to develop lung adenocarcinoma than individuals with wild-type genes (10:0 and 1:7; p=0.0003) (Table 4).

Taken together, these results indicate that the YAP1 R331W mutation is closely related to the occurrence of lung adenocarcinoma or GGO.

Example 2 Identification of YAP1 R331W Mutation and Functional Correlation of Lung Adenocarcinoma or GGO Using YAP1 Mutant Cells

In this example, the human lung adenocarcinoma cell line A549, which exhibits wild-type YAP1 , was used to study the role of the YAP1 R331W mutation in lung adenocarcinoma or GGO. ShRNA-use may be subject to the 3'UTR YAP1 YAP1 lentivirus (Lentivirus) to the silence (Silenced) derived YAP1 performance; For comparison, respectively, and would exhibit YAP1 R331W wild type YAP1 vector transfected into YAP1 - Silent A459 cells, overexpressing. The results of the experiments were evaluated using cell colony formation and cell invasion assays according to the procedures described in the Materials and Methods section. Figures 2A and 2B illustrate the results, respectively.

The results of Western blot of FIG sections 2A, compared to the control group (i.e., A549 or sh- lacZ), shRNA- YAP1 be successfully inhibited expression vector YAP protein; cytostatic (YAP1 knockdown cell performance in YAP1; In the YAP1 - wild type or YAP1 - mutant type, excessive expression of wild type YAP1 or YAP1 R331W can restore the performance of YAP1. In addition, the transfer of the YAP1 R331W mutation significantly increased the ability of YAP1 to express the cell colony forming ability of the suppressor cells, and the cell colony forming ability was even stronger than that of the control cells.

Cells violation results of FIG. 2B noted, A549 cells as compared to normal, shRNA- lacZ expression cytostatic (sh- lacZ), shRNA- YAP1 exhibit cytostatic (SH- YAP1), overexpression of exhibiting an inhibitory control vector YAP1 Cells (vectors) and YAP1 expressing inhibitory cells ( YAP1 - wild type) which overexpress wild type YAP1 , and cells expressing YAP1 R331W ( YAP1 - mutant type) in excess have strong invasive ability.

These results indicate that transduction of the YAP1 R331W mutation into the wild-type YAP1 protein increases the ability of cells to colonize and invade cells.

To summarize the above, the results of these examples indicate that the YAP1 R331W mutation may cause cell colony formation and invasion of cancer cells; the results are consistent with the results of clinical trials, that is, the YAP1 R331W mutation is hereditary and closely related to the occurrence of lung cancer. Therefore, the YAP1 R331W point mutation can be used as an indicator to assess the risk of lung cancer in a body.

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

<110> He Bingqing Yu Songliang Zhang Songyu Li Kezhao Yang Yuchi Chen Yuyu

<120> Methods and kits for detecting lung cancer

<130> P2806-TW

<160> 2

<170> BiSSAP 1.3

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> YAP1-F

<400> 1

<210> 2

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> YAP1-R

<400> 2

Claims (11)

A method for assessing whether a subject has lung adenocarcinoma or a risk of developing lung adenocarcinoma by using a biological specimen of one body, comprising: extracting DNA from the biological sample; and detecting whether the extracted DNA has a s193100333 single nucleotide polymorphism (SNP) locus; wherein, if the extracted DNA has the SNP locus of the rs193100333, it represents that the individual has lung adenocarcinoma or has lung adenocarcinoma risk. The method of claim 1, further comprising amplifying the extracted DNA by using a pair of primers having nucleotide sequences of SEQ ID NO: 1 and 2, respectively. The method of claim 1, wherein the SNP locus utilizes direct sequencing, primer extension, dynamic allele-specific hybridization (DASH), molecular letter Molecular beacon, SNP microarray, restriction fragment length polymorphism (RFLP), quantitative polymerase chain reaction (quantitative polymerase chain) Reaction, qPCR), flap endonuclease (FEN), single strand conformation polymorphism, temperature gradient gel electrophoresis (TGGE), denaturing high-performance liquid layer Detection by high-resolution melting of the entire amplicon or DNA mismatch-binding protein. The method of claim 3, wherein the primer extension method is a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The method of claim 1, wherein the biological sample is a skin slice, a whole blood sample, a buffy coat, a plasma sample, a serum sample, and a Urine sample or a mucus sample. The method of claim 1, wherein the individual is a Chinese. The method of claim 1, wherein the SNP locus of rs193100333 corresponds to a mutation of a YAP1 protein in which arginine at protein position 331 is substituted with tryptophan. A kit for detecting whether a biological sample of a body has a SNP locus of rs193100333, the set comprising: a pair of primers having nucleotide sequences of SEQ ID NO: 1 and 2, respectively, A polymerase chain reaction (PCR) is used to amplify a DNA fragment of the biological sample containing the SNP locus of the rs193100333; and a PCR reaction reagent. The kit of claim 8, further comprising a DNA template as a positive control, wherein the nucleotide sequence of the DNA template has the SNP locus of the rs193100333. The kit of claim 8, further comprising a DNA template as a negative control, wherein the nucleotide sequence of the DNA template does not have the SNP locus of the rs193100333. The kit of claim 8, wherein the PCR reaction reagent comprises PCR reaction enzyme, MgCl ₂ , deoxynucleotide triphosphate (dNTP), PCR reaction buffer, and double distilled water.