TWI380018B - Method and kit for predicting response of esophageal cancer patient to chemoradiotherapy - Google Patents

Description

1380018 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates generally to genetic markers, and more particularly to single nucleotides related to the response of a cancer patient to chemical and radiotherapy. Polymorphism ) ° [Prior Art] (4) It has become the sixth leading cause of cancer death in the world and has increased. Unfortunately, most of the esophageal cancers are taken at the end of the day, and the multimodality of therapies is used to improve the resectability of the tumor and the long-term survival rate of the patients. Concurrent chemoradiation therapy (CCRT) followed by esophagogastric resection (eS〇Phag〇gastrectomy) has been widely used in current clinical practice. However, there are individual differences in the response of the bursts, which in turn affects the treatment of the CCRT, which tends to have an improved survival rate for the CCRT, but the survival rate of the patient with the main-silk response may be delayed due to treatment. reduce. Despite the fact that the red needle has a biomarker related to the response to chemotherapy (TTze parmacogenomics journal 2009; 9:202-7; Cancer Lett ' ._17, and 7^ J 2008; 123:826-30), However, reliable genetic markers have not yet been obtained. There is still a product to be listed in Table 1 for gene labeling, and it can be predicted that ECa disease is linked to two. It helps to avoid unnecessary treatment. [Summary of the invention] S3 Ming provides a kind of prediction for the response of esophageal cancer patients to chemical and radiotherapy. a method comprising genotyping a 3 1380018 single nucleotide polymorphism (SNP) marker from a test sample taken from a patient, the marker being selected from the group consisting of rs4954256, rs16863886, and combinations thereof, wherein The presence of the C allele of rs4954256, the presence of the allele of rsl6863886, or the presence of both is an indicator of an increased likelihood of complete response to chemical and radiotherapy. ~ In another aspect, the invention provides A kit for performing the methods described herein comprising one or more polynucleotides for genotyping for rs4954256, rsl6863886, or a combination thereof. In a specific example, the kit includes Rsl6863886 performs a first set of isolated polynucleotides for genotyping. In another specific example, the set includes a genotyping assay for rs4954256 The various embodiments of the present invention are described in detail below. Other features of the present invention will be apparent from the following detailed description of various embodiments and the scope of the claims. The invention is to be construed as being limited by the scope of the invention, and the invention is intended to be [Embodiment] The present invention provides two SNP markers, rs4954256 and rsi6863886, which are identified by a two-stage whole-genome correlation study (GWAS), which have significant CCRT responses to patients with ECa. Relevant, and can provide a high degree of predictive accuracy. ^ Unless otherwise defined, 'all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In the middle, the word "一" refers to one or more (that is, at least i) one of the accepted words in the lexical grammar. The term "polynucleotide", "nucleic acid", or "nucleic acid molecule" as used herein refers to a polymer consisting of a nuclear acid unit, including naturally occurring nucleic acids, such as deoxyribose. Nucleic acids ("DNA") and ribonucleic acid ("rna"), as well as nucleic acid analogs, including those having non-naturally occurring nucleotides. Polynucleotides can be produced synthetically, for example, using an automated DNA synthesizer. The term "nucleic acid" or "nucleic acid molecule" generally refers to a large polynucleotide. It is clear that when a nucleic acid fragment is expressed by a DNA sequence (ie, a, T, G, C), it also includes an RNA sequence (ie, a, U, G, C), and a "U" in the bean. Replace "T". ”

The term "separation" as used in the text refers to nucleic acids (such as DNA or coffee) and refers to molecules separated from other DNAs or RNAs present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid that is substantially free of cellular material, viral material, or culture medium when prepared by recombinant DNA techniques, or substantially free of chemical precursor molecules or other chemicals when chemically synthesized = Nucleic acid. Meanwhile, "isolated nucleic acid" is intended to include a nucleic acid fragment which is not in a fragment form in a natural state and which is not found in a natural state. The "allele" used in the context of the invention refers to a variant of the nucleotide sequence. Bis, etc., bieiieiic p〇iymorphism has two forms. In the case of an allelic form, the diploid organism can be a homotype (h〇m〇zyg〇us) or a heterotype (heterozygous).

The term "SNP" as used herein refers to a single nucleotide polymorphism in DNA. SNPs usually have highly conserved sequences before and after, which vary only in population members of 1/1〇〇 or 1/1000. For alleles at each SNp position, the individual can be a homotypic combination or a heterotypic combination. In some cases, the SNP may be referred to as "cSNP", indicating that the nucleotide sequence containing the SNp is an amino acid "encoding" sequence. SNPs can be produced by the substitution of a nucleotide at a polymorphic site by another nucleotide acid. Substitutions can be either a permutation or a transversion. A homologous substitution is the substitution of one nucleotide by another, or the substitution of one pyrimidine by another. Heterogeneous substitutions are replaced by one mouth' or vice versa. For example, if a member of a bifamily has adenine (A) at a particular chromosomal location and another member of the population has cytosine (C) at the same position, then the position is a 5 SNP. The SNP-tagged alleles described herein can be clearly represented by bases A, c, G, or τ when jE is found in the polymorphic site in the analysis used. The naming of SNPs described in this article is based on the National Center for Biotechnological Information (NCBI), which is the official (4) SNp (5) still identifying mark for each unique SNP. The public can query the database at TM. The term "genotype identification" as used herein refers to the identification of a gene located in an individual or a sample. The "genotype 妒 =" for gene labeling of a sample or individual may include alleles on the SNPs of the sputum: for example, a specific nucleotide in a genome is in A, and In other individuals it is c. An individual having A at this position has a ^= gene, and an individual having C has a C allele. In a diploid organism, the individual has two sequence genes containing the polymorphic position and a C allele, or two equal C genes, or two C alleles. In a particular population, each allele can exist at a different frequency. There are two copies of cf=same frequency, Zhao; but two copies! I because ΐ, body 疋Α alleles are the same face, the genotype is Μ; another individual with one allele is a heterotypic combination The genotype is Ac. The term "chem 〇 radi 〇 radi radi chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem chem Therapy ir:mchem〇radiationtherapy,cCRT)" is used interchangeably here, and it is combined with therapy and radiation therapy. - "Complete response" to ccrt refers to complete remission of the tumor = no detectable symptoms, such as microscopic s, tumor, grossly visible residual tumor (tumor) ' or tumor The process. The term "bowtail" refers to a specific oligo-sequence that is complementary to the column' and is used to hybridize to the target nucleotide sequence. Primer is the starting point for the polymerization of nucleotides catalyzed by polymerase, octapolymerase, or reverse transcriptase. The term "probe" as used in iff refers to a defined nucleic acid fragment (or a nuclear fragment, such as a polynuclear collision as defined herein), which can be used to = the presence of a specific impurity of the towel, and the presence of miscellaneous acids. A nucleotide sequence that is complementary to a particular polynucleotide sequence to be recognized is included. In one aspect, the invention provides a method for predicting the response of an esophageal cancer patient to chemical and radiotherapy, comprising genotyping a SNP marker for a test sample taken from a patient selected from the group consisting of rs4954256, rsl6863886' A group consisting of a combination of the presence of the c allele of rs4954256, the presence of the G allele of rsl6863886, or the presence of both is an indication of an increased likelihood of complete response to chemical and radiotherapy. Table 1 shows the naturally occurring nucleotide sequence (SEqidn®: 1) containing rs4954256 and the nucleotide sequence containing rsl_3886 (SEQ IDN〇: 2), taken from the NCBI database (human, 々ο(10)5^, ie ^). The nucleotides in parentheses in the sequence are polymorphic nucleotides. The sequence of the 丨4954256 polymorphic nucleotide sequence is located at position 27 of SEq ID N〇:丨, while the polymorphic nucleotide of rsl6863886 is located at position 27 of seq ID NO: 2. Table 1 nucleotide sequence rs4954256 atattggagagttaacagagaatgcc[C/T]aaaactggaaaaacaaaaacttcaa (SEQIDNO: 1) rs 16863 886 aatggtgtcccttgaaggctatctgt[C/T]tgcttttggataaaatggacagaag (SEQ ID NO: 2) SNP rs4954256 is located in chromosome 2q21.3 It belongs to the sub-family of SMARTAL1. The scorpion contains a helix ^ 1380018 ΑΪΓ γ) ’ ί followed by a zinc 相关 associated with the Ran G protein binding protein. (4). However, the function of the tortoise is not clear. The SNP says that 6 is between 1 and ❼ and young on chromosome ^36.1. Catalytic a propyl thiopurine synthase Ρ: owing unit, which is a regulatory subunit that forms a tetramer with two humans. ^ Code S1P (God, i sphingosine pot acid ester) _ specific phosphohydrolase, which can be phosphoric acid, becomes sphingosine. Both the household 7 and the ΖΛ4 are related to the function of the G protein. The test sample in the 物 + substance = Yuming method may be any of the esophageal cancer patients, and the nucleic acid molecule 'includes a part of the gene sequence to be tested. Since $ = can be a sample of cells, tissues, or organs, or can be biological material like blood, milk, tears 'saliva, hair, skin, tissue, or u. The nucleic acid sample which can be used to carry out the method of the present invention may be a DNA or a genomic progenitor or an amplification product thereof, and a specific example of the test sample of the present invention is a blood sample. In the case of the invention, the method of the present invention is carried out by comparing the genotype of the test for esophageal cancer patients, wherein the _ mark is capable of being completely responsive to chemical and radioactive green therapy. Frequency = towel, the method of the present invention is carried out by the identification of the base of the esophageal cancer patient, wherein the existence of the G #丝目 is an indicator of the increased likelihood of t-study . The method of the present invention is based on the genotype identification of the esophageal cancer patient=test sample, like the combination of s4954256 and lake 6鸠, and the C allele of the ^i rs4954256 and The sl6863886 G and the like are all electrically reactive, and the patient who is evaluated for the possibility of a complete reaction to the chemical and radiotherapy according to the method of the present invention is a human adult having esophageal cancer. In general, 8 diseases are ^ γ years old or older, but under 70 years old. In some specific examples, i is divided into 1 body, 2 or 2 years old, but under 70 years old. In addition, $Japan; "Γ" The patient described in this article is an Asian patient, especially a Chinese sputum. In some specific cases, the patient is a male. ίίίί is known to have a shirt method Example of a sample SNP specific SNP can be used - or multi-turn nuclear _ probe or primer, amplifying acid pair of library rc nuclear Wei hybrid amplification primer pair, the target polynuclear position. Can be used to implement the method of the present invention The nucleus of the if part, which includes the position of the SNP, and the presence of the SNP in the sputum can be selected by the probe. This method can further include making the target polynuclear. The thief thief contacts the endonuclease and detects the cleavage of the probe, which is determined by whether the nucleotide acid present in the SNp site is complementary to the corresponding nucleotide of the probe. a pair of probes that are present at the polymorphic position of the county (4) at a polymorphic position and adjacent to the upstream position of the site and the downstream heterozygous position, and wherein one of the probes includes f ^SNP exists in the nuclear interaction _ terminal acid. When the terminal of the Cong pin nucleoside acid and exist When the riboic acid is complementary, the selective hybridization may include the terminal nucleotide acid'. In the bonding enemy, the upstream and lower cores may be joined by bitter acid. Thus, the presence or absence of the bonding product may be the SNp. An indicator of the presence of a site nucleotide. One of the examples of this type of analysis is the system (Applied Biosystems, Foster City, CA). Oligonucleotides can also be used as primers, for example, primer extension reactions, wherein the extension reaction The product (or no product) is an indicator of the presence of the nuclear crane. Further, a primer pair for amplifying a portion of the target polynucleotide including the SNP site can be used, wherein the amplification product can be examined to determine the SNp site. The nucleotides are present. Here, useful methods include the 1380018 method which can be easily changed to a high throughput format, a multiple form (mukipiexf〇rmat), or both. The primer extension or amplification product can be used. Various methods known in the art are directly or indirectly detected and/or sequenced. Traditional sequence methods can be used to sequence amplification products encompassing SNPs, such as dideoxy chain termination (di Deoxy-mediated chain termination method), also known as the Sanger Method, and the chemical degradation method, also known as the Maxam-Gilbert Method. The present invention can be used in this technique. Known to analyze flux-to-high-throughput systems in SNPs, such as the Mass ArrayTM system (Sequenom, San Diego, CA), BeadArrayTM SNP genotyping system (San Dieg〇, cA), 'and Affymetrix GeneChip® Human Mapping 500K array (Affymetrix, Inc" Santa Clara, CA). The SNP detection method used to practice the present invention generally utilizes selective impurities. As used herein, the term "selective hybridization" or the like refers to hybridization under moderately stringent or highly stringent conditions such that a nucleotide sequence will be biased to bind to a selected nucleotide sequence (compared to In the case of unrelated nucleotide sequences, the degree of bias is large enough to identify the presence of nucleotides in SNp. It is clear that a certain degree of non-specific hybridization is unavoidable, but as long as the hybridization of the target nucleotide sequence is sufficiently selective, it can be distinguished from the non-specific hybridization side. , the non-specific second, for example, at least about 2 times higher selectivity, generally at least ^ more selective selectivity 'usually at least about 5 times higher selectivity, specifically at least about 'paste' The above selection is (10) labeled oligo-oligo acid and its relative hybridization sequence. The relative GC.AT bath is the parent; JJ: Xi Xixi E r*r is) 1380018

Laboratory Press, Cold Spring Harbor, Ν.Υ.; and Ausubel et al., 1998 'Modern operational procedures for biomolecular ^^ plus M?/ecw/izr calendar J. Wiley & Sons Inc., New York, 1988. In general, stringent conditions are about 5-30 ° C below the specified melting point (Tm) at a given ionic force and pH. Or 'strict conditions are about 5-15 〇c below the Tm of the specified sequence at the specified ion and pH. For example, a strict heterogeneous condition has a strontium salt concentration of less than about 1.0 bismuth (or other salt) ion concentration, typically from about 0.01 to about 1 Μ sodium ion concentration, at about ρ Η 7.0 to about ρ 8.3 8.3, and the temperature is short. The probe (eg, 10 to 50 nucleotides) is at least about 25 〇c, and for long probes (eg, greater than 50 nucleuses) at least about 55. (^. Exemplary non-strict or low-degree conditions for long probes (eg, greater than 50 nucleotides) may include 20 mM Tris, pH 8.5, 50 mM KC1, and 2 mM MgCl 2 buffer And 25. The reaction temperature. According to the present invention, the method described herein can determine whether the esophageal cancer patient is more likely to have a complete response to chemical and radiotherapy based on the genotype of rs4954256 and/or rsl6863886. It is shown that the c allele of rs4954256 and the G allele of rsl6863886 are protective alleles; compared with the patient population that does not fully respond to chemical and radiotherapy, the f-gene is more common in a disease that is completely responsive to chemotherapy and radiation therapy

In the ethnic group, therefore, the presence of the C allele of rs4954256 or the G allele of rsi6863886 indicates an increase in the complete response of these patients to chemical and radiotherapy. Specifically, as the number of C alleles of rs4954256 or the G allele of rsl6863886 increases, patients are more likely to have complete responses to chemical and radiotherapy (eg, at least 15, 5, 2.0, 25, 3) 〇, 3 5, 4:0, 45, or 5.0 times chance). More specifically, with the increase in the number of C alleles of rs4954256, the patient has a 4.54 chance to fully respond to chemotherapy and radiotherapy; and with the increase in the number of G alleles of rsl6863886, patients There is a 3.84 times chance to fully respond to chemical and radiotherapy. ”

[S 1380018 Isolated polynucleotides, for example, can be used as primers or probes for genotyping of SNPs of the invention, wherein such polynuclei can be readily determined based on the information of the SNps and related nucleic acid sequences provided herein. The sequence of the nucleotide. A variety of computer programs, such as SeqTool Document vl.0 (IBMS, Taiwan), can be used to quickly get the best primer/probe set. In a specific example, genotyping of rs4954256 is performed using a first primer pair having fEQ ID NOS: 3 and 4, respectively. In another specific example, genotyping of rsl6863886 is performed using a second primer pair, which has SEqID NOS: 5 and 6, respectively. Table 2 shows the sequences of these primers.

Rs4954256 forward primer 5'- ACGTTGGATGTCTACCGTTTCCCGTATCTC-3' (SEQ ID NO: 3) 3,- ACGTTGGATGCCATATTGGAGAGTTAACAG-5, (SEQ ID NO: 4)

Reverse primer rs!6863886 Forward primer Reverse primer 5 -ACGTTGGATGCTGCTTAAGGCAATGGTGTC-35 (SEQ ID NO: 5) 3,- ACGTTGGATGTTACTTTGGCCCTTCTGTCC-5, (SEQ ID NO: 6)

In another aspect, the invention provides a φ reading kit for performing the methods described herein, comprising one or more isolated polynucleosides for genotyping identification of rs4954256, rsl6863886, or a combination thereof acid. Specifically, the isolated polynuclear thieves serve as primers or probes for performing the SNPs of the present invention. 1 ^ In some specific examples, the sets are PCR sets. In an example, . The kits include: (a) to amplify the primers described herein; and (b) buffers and enzymes 'including DNA polymerases. Part of the specific example order, these sets are microarray sets. These sets are one after another. A probe attached to a solid support surface. These probes can be described by Detective 12 (§) 1380018. In a specific embodiment, the probes are referred to herein, 苴#. These kits may also contain hybridization reagents and/or reaction skimmers. The signal generated by the two furnaces while hybridizing with the target nucleic acid sequence is examined. Further, the materials and reagents of the microarray kits are placed in one or more groups, and the components are generally individually placed in an appropriate manner, and can be easily designed and synthesized in the present invention for the nucleic acid region of interest. The appropriate primer or probe can be used to make t: ;, ; 8^9:^ 2 2pL2, 3 days, or 25 nuclear thieves. Although the maximum length of the probe can be the same as the sequence (depending on the type of analysis used), -2, ^ = about 50, 60, 65, or 7 核 nucleolic acid. As far as the primer is concerned, at least about 30 cores are acid at 22 degrees. In a specific example, Ιιΐίΐίΐϋ is between about 18 and about 28 nuclear thieves. However, for example, nucleic acid arrays and other probes are immobilized at the base length, such as the 'length of about, is, the gene two has the primers of SEQ ID: 3 and 4. Group separated polynuclear. Specifically, the separation & S, the introduction of positions 5 and 6. In yet another example, the second group comprises the first and second sets of isolated polynucleotides described above. ^本,明' The kit can further include the polymorphisms of the genes, and (4), such as, (1) reagents for purifying nucleic acids; 1380018 dNTPs, depending on the situation, one or more special Labeled dNTps; (3) Synthetic labeling reagents such as chemically activated derivatives of fluorescent dyes; (4) enzymes, various =, reverse transcriptase, DNA polymerase, and the like; (5) various buffer matrices 'eg, hybridization and washing buffers; (6) purification reagents and components of labeled probes, eg, centrifuge tubes, etc.; and (7) signal generation and detection reagents, eg, streptavidin-base Phytase conjugates and the like. > In some examples, the kit of the present invention contains instructions for the results obtained by the SNPs. Specifically, the description of the instructions, genotype identification results t, the presence of the C allele of rs4954256 and the rsl6863886 allele are indicators of an increase in the complete response to chemical neoplasm therapy. More specifically, the narrative of the narrative, the number of c alleles of P-remaining ,, will be able to assess the disease of about 4.54: complete response to chemical and radiotherapy, and the number of rsl6863886 increases 'It will be able to assess the patient's chance of a 3.84-fold chance to fully respond to chemugin k radiotherapy. Various specific examples of the invention. Other features of the invention will be apparent from the detailed description of the embodiments and the appended claims. Example 1: Patient population and therapy The study included 90 patients with ECa, male, less than 7〇巅, for further esophagectomy. The patient ί ί 匕 匕 匕 匕 匕 匕 匕 匕 放射 放射 放射 放射 放射 放射 放射 放射 放射 放射Surgery around! Blood cells: Take a peripheral blood sample. Separation and storage, uranium-based treatment (on the first day of the week and the first day of the week, 1 note, urine money (225 m_2 per day) and / or too much radiation ^: ^ =% = 35 — _cGy After the front field technology. =^ Use the standard before and after / and according to ~ lung correction, nutritional status, and general performance state with a patient who can accept ^ 14 丄JOUU丄s ϋί' for esophageal peak surgery and stomach According to the pathological evaluation of the surgical specimen, the silk patient will receive a complete response to complete remission of CCRT string and sputum, and an adverse reaction to observe the progress of the disease. Table 3 summarizes the 90

Age (year:) average SD smoking

55.20 7.2836 54.87 8.2263 No No data Drinking No information Chewable 摈榔 No data 32 7 5 31 6 9 32 7 5 31 6 9 3 6 12 5 12259 Example 2: Two-stage whole-genome correlation study (GWAS) ^ A two-stage GWAS was performed to identify the CCRT for predicting ECa patients. Figure 1 shows the schematic flow of the study design. Phase 1 =2006; 38:209-13 suggested that Phase 1 includes approximately 30% f. Therefore, 15 patients were randomly selected from CCRT complete responders (η=44) and no responders (η=46). Table 4 summarizes the clinical features of these 30 patients and the remaining 6 patients. S S1 15

Age (years) Average SD No data available Drinking is 55.67 6.2526 54.6 7.8944 54.97 7.8580 55 8.5049 No No information on chewing betel nut No data 11 2 2 11 2 2 4 9 2 10 3 2 10 3 2 5 8 2 21 5 3 21 5 3 9 17 3 21 3 7 21 3 7 7 17 7 Protein imitation extraction ' 再 · 5% SDS and 200 - restriction enzymes (Nspi or Stvn Stroke genomic DNA. To make it digestively digested, then ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; A DNA fragment containing an adapter, such as a universal field, which recognizes an adaptor sequence, is used. The PCR device was subjected to a process of amplifying a fragment ranging from 200 to 丨 in a size range of 1 (10) bp. Then, the amplified DNA was subjected to m-type, labeling, and interfering with the GeneChip® Hu^ian Mapping 5〇〇κ array (Affymetrix, heart, blood for 14 hours at 49 C, and then the array was cleaned with Fluidics Station 450. Scanning was performed with the GeneChip Scanner 3000. The correlation between individual SNPs and CCRT responses was explored using Fisher's exact test. According to the following two criteria: (丨) has at least three in a defined genomic region Strict values (8) 丨 continuous SNPs; and 1380018 (2) the most significant SNPs in this region (/V〇y/a 2006; 66: 1556-64), 26 candidate SNPs were obtained in phase 1. Stage 2

The genotypes of 26 candidate SNPs were further confirmed for all 9 patients according to the iPLEX protocol [using the MassARRAY system from Sequenom (San Diego, USA). This analysis was carried out based on the adhesion reaction produced by the primer adjacent to the polymorphic site. PCR primers and extension primers were designed using the software SeqTool Document vl.0 (IBMS, Taiwan). The addition of a DNA polymerase plus a mixture of nucleotides and a terminator allows the primer to be extended through a polymorphic site' and produces a product of a particular mass. The mass of the primer extension product was then analyzed using MassARRAY TyperAnalyzerv3.3 software (Sequenom) to determine the nucleotide sequence of the polymorphic site. For the two SNPs, rs4954256 and rsl6863886, the following primers were designed for PCR amplification: for

S'-ACGTTGGATGTCTACCGmTCXrCX}!1 ATCTC-3' and 3'-ACGTTGGATGCCATATTGGAGAGTTAAC AG-5' used for rs4954256; and 5,-ACGTTGGATGCTGC TTAAGGCAATGGTGTC-3' and 3'-ACGTTGGATGTTACT TTGGCCCTTCTGTCC-5' for rsl6863886. For the two SNPs, the following PCR conditions were used: 0.5 mM each primer, 2 mM mM dNTP, 2.5 units of Taq polymerase, standard polymerase buffer containing enzyme (15 MgC12), and 150 ng of genomic DNA. The total volume of the PCR mixture was 25 ml. The pcr temperature program is: 95. (: denaturation for 5 minutes; 95 ° C for 1 minute for 35 cycles, 55 ° C for 1 to 75 minutes, and 72 ° C for 1.75 minutes; and 72 ° C for the end of 1 minute. Use 6% agarose gel to 5 〇w electrophoresis of the PCR product for 30 minutes. Using MassARRAY TyperAnalyzer v3.3 software (Sequenom,

San Diego, USA), to manually determine the classification of SNPs. An independent Fisher accuracy test is performed on the remaining 6 samples (outside the 1st and 30th samples). No significant differences were observed between the two populations (data not shown). Therefore, the collection is taken from the data of all 90 samples to obtain the enthalpy in phase 2. Ί- ώ· Statistical power. In both stages i and 2, there was no significant difference in mean age between complete responders and adverse responders (data not shown). In addition, clinical characteristics (including smoking, alcohol consumption, and chewing betel nut) were not significantly different between complete responders and adverse responders and were therefore not included in further analysis (data not shown). The genotypes of 30 patients in P white 1 were confirmed by & 〇 〇 111 data; 8 SNPs with high replication error rate and low interpretation rate were excluded from further analysis. The sample in Phase 2 consisted of the 30 patients and the remaining 6 patients. In the case of P white and 2, the problem of multiple experiments was solved using the B〇nferr〇ni correction. After calibration, the additive model showed two SNPs, rs4954256 and rsi6863886, which were significantly associated with CCRT response (rs4954256: OR=3.84, 95% CI=1.56-9.43, value=〇4〇02; rsl6863886: OR = 4.54,950/〇ci=l.81-11.40, value=9χ 104). Table 5 lists the statistics of 18 candidate SNPs in stages i and 2. 1380018 Table 5 Stage 1» Stage Γ ·* (λ=90) Minor 0=30) MAF5 Alleles Completely Poor

SNP position gene gene corpse-value 55 responder responder p-value 55 OR (95% CI) 2 rs 12713098 2pl6.3 XRXN1 A 7.64 xlO'3 0.500 0.3667 0.092 1.66 (0.87-3.17) rs4954256 2q21.3 ZRANB3 C 3.85 X10'5 0.2841 0.0930 0.002 3.84 (1.56-9.43) rsl6863886 2q36,l Intergenic G 4.75xl0'7 0.2841 0.0870 9x1ο-4 4.54 (1.81-11.40) rs4284824 2q37.1 INPP5D C 5.35X10-5 0.3182 0.4651 0.062 0.54 (0.29 -1.01) rs4697204 4pl5.31 Intergenic G 1.02X10·4 0.4886 0.3256 0.032 1.98 (1.05-3.74) rsl876266 4pl6.1 Intergene V 1.70X10·4 0.3068 0.1413 0.012 2.88 ( 1.30-6.37) rsl 440971 7q32.1 Intergenic C 1.73x1 O'5 0.2045 0.1047 0.093 1.99 (0.88-4.54) rsl630140 9q22.2 Intergenic C 1.31X10-4 0.1364 0.3023 0.010 0.32 (0.14-0.74) rsl805740 12p31.13 PHC1 C 6.31X10"4 0.2955 0.2093 0.224 1.53 ( 0.78-3.00) rs4240039 Xpll.4 Intergene G 7.97XHT4 0.1364 0.2558 0.057 0.68 (0.39-1.17) rs4830776 Xp22.2 Intergenic C 1.23X10·4 0.2045 0.3571 0.028 0.68 (0.42-1.10) rs5937044 Xql3.1 Intergenic A 1.61 X10·5 0.2954 0.2791 0.868 1.04 (0.6 5-1.66) rs927142 Xq21.31 Intergenic G 1.55xlO'5 0.5 0.444 1 1.12 (0.73-1.85) rs5990542 Xq2l.33 Intergenic C 1.31X10-4 0.5227 0.4286 0.226 1.21 (0.79-1.85) rs5910842 Xq24 Intergenic A 3.56 X1 O'7 0.5 0.3043 0.010 1.41 (0.92-2.15) rsl0521750 Xq25 Intergenic C 1.91X10'6 0.5682 0.3810 0.015 1.46 (0.95-2.25) rs5951775 Xq27.3 Intergenic T 1.68xl〇·5 0.1591 0.0698 0.095 1.59 (0.78- 3.24) rsl 202918 Xq28 intergene A 4.53xl0'5 0.5114 0.3478 0.035 1.41 (0.92-2.15) § MAF represents the minor allele frequency so that the p-value is taken from Fisher's Accuracy check*p-value based on use Data calculated from 30 patients from 30 patients** The results in Phase 2 were calculated from the total of .90 samples from the mass spectrometry and the rabbit OR represents the odds ratio and is calculated using the additive model [S1 19 1380018 In addition to the Fisher's fine test, the C〇chran-Armitage trend check was also performed to confirm that rs4954256 (ρ·value=0.002) and rsl6863886 (p-value=6xl0~) increased with the number of minor alleles. 4) Significantly correlated with ccRT response. With the increase in the number of minor alleles, SNP rsl6863886 was significantly associated with a full CCRT response of 4.54x chance (95% confidence interval (CI) = 1.81-11.40); whereas SNPrs4954256 had a full CCRT response with a 3.84x chance Significant correlation (95% CI=1.56-9.43). In addition, according to the additive model, the correct rate of the Leave-One-Out Cross Validation (LOOCV) used to predict the CCRT response was 64.37% (56 of 87 patients) for rs4954256, and For

Rsl6863886 is 68.89% (62 out of 90 patients). Combining the two SNPs resulted in an increase in predictive accuracy to 72% (63 of 87 patients). The sensitivity of this experiment (correct prediction of non-responders) and specificity (correctly predicted responders) were 70% and 75%, respectively. The positive predictive value was 71% and the negative predictive value was 73%. The regression coefficients of rs4954256 and rsl6863886 are 1·=72 and 1.5745, respectively, indicating that the probability of a complete CCRT response increases as the number of minor alleles increases. These results indicate that rs4954256 and rsl6863886 are strongly associated with CCRT responses in patients with ECa and can be used to predict CCRT responses.

This first phase of GWAS identified a CCRT response with high accuracy rates to predict treatment of ECa. Two snps, =495C56 and rsl6863886 were found here, which were significantly associated with complete CCRT response (along with the increase in the number of alleles) and provided a high level of predictive accuracy ^7^41%) The gene polymorphism determined by the blood sample does not, and the time change 'is relatively stable relative to the body mutation taken from the tumor tissue, which changes with the progress of the disease. The two SN?s described in the present invention can be used to measure the response of an ECa patient to CCRT, and thus the most appropriate treatment for the patient can be determined based on the predicted results. BRIEF DESCRIPTION OF THE DRAWINGS [0007] The foregoing summary, as well as the foregoing description of the embodiments of the invention, For the purposes of the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the preferred embodiments shown. In the drawings: Figure 1 shows the outline flow of the example study design above.

[S3 21 1380018 Sequence Listing <110> National Taiwan University <120> Biomarker for predicting the response of esophageal cancer patients to chemical and radiotherapy <130>, 0006TW <160> 6 <170> Patentln 3.5

<210> 1 <211> 52 <212> DNA <213> Human (Homo sapiens) <220> <221> <222> (1) _· (52) <400> 1 atattggaga Gttaacagag aatgccyaaa actggaaaaa caaaaacttc aa 52

<210> 2 <211> 52 <212> DNA <213> Human (Homo sapiens) <220><221><222> (1) .. (52) <400> 2 aatggtgtcc Cttgaaggct atctgtytgc ttttggataa aatggacaga ag 52 <210> 3 1380018 <211> 30 <212> DNA <213>Artificial sequence<220><223> rs4954256 primer<400> 3 acgttggatg tctaccgt11 cccgtatctc <210 〉 4 <211> 30 <212> DNA <213> Artificial sequence <220><223> rs4954256 primer <400> 4 gacaattgag aggttatacc gtaggttgca <210> 5 <211> 30 <212&gt DNA <213>Artificial sequence<220><223> rs 16863886 Introduction <400> 5 acgttggatg ctgcttaagg caatggtgtc <210> 6 <211> 30 <212> DNA <213> Artificial sequence <;220><223>rs 16863886 Introduction 1380018 <400> 6 cctgtcttcc cggtttcatt gtaggttgca 30

Claims (1)

Patent No. 098,135, 482, 2, 4, 1 / or a method of reaction of paclitaxel in combination with radiation therapy, comprising a test sample of two for a single nucleotide polymorphism (SNP)-tagged gene g 疋 'the marker is selected from rs4954256, rsl6863886, and combinations thereof The group consisting of the presence of the C allele of rs4954256 (located at position 27 of SEq), the presence of the rsl686388RG allele (located at position 27 of seqidn〇2), or the presence of both is for the therapy An indicator of the increased likelihood of complete response. ~, -2. According to the method of claim ,, which comprises genotyping the rs4954256 of the test sample taken from the patient, wherein the presence of the c allele of the SNP marker is fully responsive to the therapy An indicator of increased likelihood. - 3. According to the method of claim, comprising the genotyping of rsl6863886 for a test sample taken from the patient, wherein the presence of the g allele of the SNp marker is a complete response to the therapy An indicator of increased sex. 4. The method of claim 1, comprising genotyping the combination of rs4954256 and rsl6863886 for the test sample taken from the patient, wherein the C allele of rs4954256 and the G allele of rs16863886 are present/existent It is an indicator of an increased likelihood of complete response to the therapy. 5. The method according to claim 1, wherein the genotype identification of rs4954256 is performed using the first primer pair. The first primer pair has SEQ ID N〇s: 3 and 4, respectively. 6. According to the method of claim 1, wherein the genotype identification of rsl6863886 is performed using a second primer pair, and the second primer pair has seqidn〇s: 5 and 6 〇7, respectively, for implementing the method according to claim 1. a set comprising a first set of isolated polynucleotides for genotyping for rs4954256, comprising primers having SEQ ID NOS: 3 and 4, respectively; for carrying out the invention patent No. 0981354482 for !^6863886 Replacement of the patent application scope without a slash after the application is amended (August 27, 2011) The second group of isolated polynucleotides identified by genotype, including SEQH) NOS: 5 and 6, respectively Primer; or a combination thereof. 8 to a kit according to claim 7, which comprises a first set of isolated polynucleotides for genetic identification of rs4954256, which comprises primers having NOS: 3 and 4, respectively. - 9. The kit according to claim 7, comprising a second set of isolated polynucleotides for base-type identification of rsl6863886, comprising primers having SEq Π) * NOS: 5 and 6, respectively . · 10. The set according to claim 7 which comprises a first set of isolated polynucleotides for base dust type identification of rs4954256, comprising primers having SEqID Snow I NOS: 3 and 4, respectively, and A second set of isolated polynucleotides for genotyping of rsl6863886 'includes primers with SEq ID N〇s: 5 and 6, respectively. 11. The kit according to claim 7 which further comprises instructions for use, wherein the result of genotypic identification is the C allele of rs4954256 (located at position 27 of SEQ ID NO: 1) and/or after 1*516863886, etc. The presence of a locus (located at 8 ugly (31): position 2 of 2) is an indicator of an increased likelihood of complete response to the therapy.