KR20150121869A - Method of Selecting an anti-cancer drug for loading on an anti-cancer patch - Google Patents

Abstract

The present invention relates to a gene marker for prognosis after a surgery and selection of an anticancer agent. More particularly, the present invention relates to a K-ras gene or a DPC4 gene marker for observing prognosis after a surgery on a patient with pancreas cancer by using genetic mutation and for selecting an anticancer agent proper for each genetic mutation. A method for observing prognosis after a pancreas cancer surgery based on mutation of the K-ras gene and an expression amount of DPC4 is based on analysis of genetic mutation and a survival rate with respect to a large number of Korean pancreas cancer patients. The method has high reliability of an experiment and analysis and thus can be advantageously used to determine whether a pancreas cancer surgery should be performed or not. If a reaction mechanism of the genes is considered, the method is expected to provide information useful to select a proper anticancer agent and to determine an injection amount and an injection concentration when an anticancer agent is injected after a pancreas cancer surgery.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of selecting an anti-cancer drug for a cancer-resistant patch,

The present invention relates to a method of selecting an anticancer agent for the anticancer patch, and more particularly, to a gene marker for diagnosing an operation prognosis and selecting an anticancer agent, and a method of selecting an anticancer agent for preventing recurrence or metastasis after surgery for pancreatic cancer. More specifically, K- ras was used to diagnose the prognosis of pancreatic cancer patients through genetic variation and to select appropriate anticancer agents for each genetic variation Gene or DPC4 gene marker and a method for providing information for selecting and / or determining the dose of an anticancer drug loaded on an anticancer patch for preventing recurrence or metastasis after surgery for pancreatic cancer.

Pancreatic cancer is notorious for aggressive local invasion and early metastasis, bad outcomes, and is the fourth highest cancer death in the United States. In 2011, 44,030 patients were diagnosed with pancreatic cancer in the United States and 37,660 patients were estimated to die from it. In Korea, pancreatic cancer accounts for 2.7% of all cancers and their 5-year survival rate is only 7.3% Do. In 2002, the mortality rate of pancreatic cancer in males and females was 96.4% and 91.6%, respectively. The high rate of mortality in pancreatic cancer was attributed to only a small number of patients diagnosed at the early stage of cancer, Or distal pancreatectomy, and the 5-year survival rate after resection of these patients is only 9 to 21%.

Development and growth of pancreatic cancer is caused by a variety of genetic variations such as activation of oncogenes, loss of tumor suppressor gene function, and overexpression of the receptor-ligand system. Among these genetic mutations, genes such as K- ras , p53, c-erbB-2 and CPC4 have been reported to play an important role in pancreatic cancer development and progression. Mutation-induced activation of K- ras tumor genes has been reported in 47-100 % of pancreatic ductal cell carcinomas and K- ras mutation has been reported to be associated with all stages of pancreatic cancer as well as benign pancreatic disease and pancreatic duct degeneration . On the other hand, gene deletion or mutation of the p53 tumor suppressor gene is very common in pancreatic cancer (40-87%), and p53 mutant forms can accumulate in the cell nucleus. In addition, overexpression of the c-erbB-2 oncogene in 20-80% of the various types of tumors and pancreatic ductal cell carcinoma is associated with invasion and poor prognosis in most cancers. The DPC4 gene, which is inactivated in 55-66% of pancreatic ductal cell carcinomas, is one of the major tumor suppressor genes targeted for penetration of pancreatic ductal carcinoma, but its precise role is unknown. Inhibition of DPC4 expression occurs late in tumor progression and induces development upon invasion of pancreatic cancer at the stage of histologically recognizable cancer progression. Thus, their inactivation may be specific to relatively invasive pancreatic cancer.

The present invention is based on the analysis of the correlation between these genes and the clinical characteristics and survival data of pancreatic cancer in Korean patients. In particular, the genetic variation of K- ras , p53, c-erbB-2 and DPC4 And as a marker for prognosis of recurrence or metastasis after pancreatic cancer surgery.

1. Korean Journal of Pancreatic Surgery vol.4 no.1 Volume 7 (2000. 2) pp.111-121

Accordingly, an object of the present invention is to provide a composition for diagnosing prognosis after surgery for pancreatic cancer comprising a gene marker.

It is another object of the present invention K- ras Mutation of the gene or the expression level of the DPC4 gene to provide a diagnostic method for the prognosis after surgery for pancreatic cancer.

It is another object of the present invention K- ras Gene or a nucleic acid that specifically binds to the DPC4 gene. The present invention also provides a composition for screening anticancer agent for anti-cancer patch, which is applied after surgery for pancreatic cancer.

It is another object of the present invention K- ras Mutation of the gene or the expression level of the DPC4 gene to provide information for selecting an anticancer agent for preventing recurrence or metastasis after surgery for pancreatic cancer.

In order to achieve the above object, the present invention K- ras Gene or a nucleic acid that specifically binds to the DPC4 gene.

In one embodiment of the present invention, the post-operative prognosis may be any one selected from the group consisting of metastasis, recurrence, survival and disease-free survival.

In one embodiment of the present invention, the nucleic acid may be a primer or a probe.

The present invention also relates to a method for detecting a mutation of a K-ras gene or an expression amount of a DPC4 gene in (A) a sample collected from a patient suffering from pancreatic cancer; And (B) comparing the measured value of step (A) with a reference value to predict the prognosis after surgery for pancreatic cancer.

In one embodiment of the present invention, the sample may be a tumor cell or tumor tissue.

In one embodiment of the present invention, the prognostic method for post-operative pancreatic cancer surgery is K- ras If GCT, the 12th codon of the gene, is mutated, the survival rate after pancreatic cancer surgery may be judged to be low.

In one embodiment of the present invention, the method for diagnosing a prognosis after surgery for pancreatic cancer is to determine that the survival rate of pancreatic cancer surgery is low when the GCT is mutated into any one selected from the group consisting of GAT, GTT, CGT, TGT and CTG .

In one embodiment of the present invention, the method for diagnosing the prognosis after surgery for pancreatic cancer may be that the survival rate after surgery for pancreatic cancer is low when the expression level of the DPC4 gene is decreased.

The invention also K- ras Gene or a nucleic acid that specifically binds to the DPC4 gene. The present invention also provides a composition for screening anticancer agents for anti-cancer patches applied after surgery for pancreatic cancer.

The invention also K- ras Gene or the DPC4 gene for the selection of anticancer drugs to be applied to an anticancer patch applied after surgery for pancreatic cancer.

In addition, the present invention relates to (A) a sample obtained from a pancreatic cancer patient, K- ras Determining the mutation of the gene or the expression level of the DPC4 gene; And (B) determining the type, dose, and / or concentration of the anticancer drug to be administered after the operation of the pancreatic cancer according to the measurement value of the step (A), and selecting the anticancer agent for preventing recurrence or metastasis after surgery for pancreatic cancer. Lt; RTI ID = 0.0 > information. ≪ / RTI >

Based on the mutation of the K- ras gene and the expression level of DPC4 according to the present invention, the method for diagnosing the prognosis after surgery for pancreatic cancer is based on the analysis of gene mutation and survival rate in large-scale Korean pancreatic cancer patients, Of patients with pancreatic cancer who are at high risk for pancreatic cancer surgery and considering the mechanism of action against these genes, it is useful information for determining the proper selection, dosage and concentration of anticancer drugs after pancreatic cancer surgery. .

Figure 1 is a Karplan-Meier survival curve, while A is the overall survival rate of patients inde K-ras and a mutant of the gene associated with, the 33.3% k-ras 5-year survival rate of patients with wild-type, K- ras The 5-year survival rate of the gene mutation patients was 14.7% (p = 0.001). B represents the patient survival analysis associated with the inactivation of the p53 gene (p = .999), C represents the overall survival analysis of patients associated with overexpression of the c-erbB-2 gene (p = 0.79), D represents the DPC4 gene (P = 0.047) of the patients associated with inactivation of the disease.
Figure 2 shows the Karplan-Meier survival curves. A was the overall survival rate between wild - type (GGT) and GAT subtype mutations in the K-ras gene. The 5-year survival rate of wild type and GAT mutation was 33.3% and 14.6% (P < 0.001). B, on the other hand, compared overall survival rates between wild-type and GTT subtype mutations (P = 0.342), C was the overall survival rate between patients with GTT and GAT subtype mutations (p = 0.061) D compared the overall survival rate between patients with CGT and GAT subtype mutations (p = 0.210).

Pancreatic cancer is known to be one of the leading causes of cancer mortality in most western countries, including Asia. On the other hand, patients with pancreatic cancer who were treated with surgery had only a very small number of patients diagnosed at an early stage, and the survival rate at 5 years after surgery was also reported to be very low.

On the other hand, the research on genetic variation and clinicopathologic prognosis of pancreatic cancer is very limited in Asian countries including Korea. Therefore, the present inventors have found that the correlation between gene mutation and prognosis of pancreatic cancer can be confirmed in a large- The present invention is characterized by the fact that the genetic mutation marker capable of determining the prognosis of pancreatic cancer, particularly pancreatic duct cell cancer, was first identified.

K- ras (NM_004985, NP_004976) mutations are found in pancreatic cancer as well as benign pancreatic disease, and are found in all stages of pancreatic duct degeneration. Thus, the mutation of K-ras is considered to be the initial process of pancreatic carcinogenesis, and the various subtypes of K- ras mutation frequency and mutation examined in the present invention are shown in Tables 1 and 2 below.

In the present invention, the frequency of K- ras mutation was 53.8% and the ratio of GTT K- ras mutation subtype was 14.5%. In addition, the tumor suppressor gene, p53, is induced in response to damaged DNA and induces cell cycle arrest at the G1 checkpoint. It has been reported that p53-encoding genes are frequently mutated in a variety of cancers found in humans. If the function of p53 is lost by this mutation, cell cycle arrest at the G1 checkpoint due to DNA damage is not achieved, Instead of repairing the damaged DNA, it is replicated and transferred to the daughter cells. Thus, the inventors have also studied the genetic mutation of p53. According to the present invention, the ratio of abnormal p53 in pancreatic cancer was 38.1%. On the other hand, according to the results of the present invention, the c-erbB-2 oncogene gene was overexpressed in 7.3% of the samples, which is considerably smaller than that in the Western countries. Suggesting that the product HER2 / new (ie, Herceptin) may not be appropriate for Korean patients as anticancer drugs.

Another gene associated with pancreatic cancer is DPC4 (SMAD4, NM_005359, NP_005350). DPC4 expression is known to be decreased in the later stages of tumor progression, and decreased expression of DPC4 causes pancreatic cancer infiltration. In the present invention, the reduction ratio of the DPC4 gene was 81.6%, which was much higher than that in the Western study (55-66%). As described above, the present invention, which relates to the correlation between genetic mutation and survival rate of pancreatic cancer for the first time on the basis of Korean pancreatic cancer patients, shows that the genetic mutation of Korean pancreatic cancer patients is significantly different from the genetic mutation rate .

These differences are judged to be based on genetic, geographical, and racial characteristics. In the same context, recent studies by Dong et al. Have shown that there are significant differences in mutation patterns found between Chinese and foreign pancreatic cancer patients .

On the other hand, the present inventors analyzed the relationship between the state of the four genes and the overall survival rate of the patient using tumor samples. As a result, the 5-year survival rates of the K- ras gene wild-type and mutant type patients were 33.3% and 14.7%, respectively (see FIG. 1A). In particular, the overall survival rate of the GAT subtype was significantly reduced compared to the wild type (P <0.001, see FIG. 2A). In the GAT subtype, mean survival rate was also significantly reduced compared to other mutant subtypes. Thus, it was confirmed that the GAT subtype mutation of the k- ras gene is closely related to the bad prognosis (see Table 2, Figs. 2A and 2B). In addition, the decrease in DPC4 gene expression was highly correlated with the survival rate of the patients.

Thus, the present inventors have conducted a comprehensive analysis of clinical, pathological and genetic factors affecting the overall survival rate of patients in a univariate (see Table 3) and multivariate (see Table 4) Prognostic factors. Previous studies have reported that mutation of the p53 gene may lower survival after surgery and may be a risk factor for disease-related death when the combination of three tumor suppressor genes p16, p53 and DPC4 is inactivated . However, according to the results of the present inventors' study, mutation of p53 tumor suppressor gene did not act as a bad prognostic factor in pancreatic cancer patients in Korea, and inactivation of DPC4 gene was predictive of a much worse prognosis OR, 2.166, see Table 4). On the other hand, the conventional R K- ras It has been reported that the mutation of the gene alone is not correlated with the survival rate of pancreatic cancer patients, but according to the results of the present inventors, K- ras Mutation alone was found to correlate with patient survival (OR, 1.631, Table 4), and among the mutations, the GAT subtype was strongly correlated with patient survival. Although DPC4 and K- ras The DPC4 gene was found to be more frequent in the Korean patients (81.6% vs 55-66%) than in the Western patients, and the DPC4 gene was found in the K- ras gene But the prognostic factors were not significantly different.

Taken together, the present inventors conducted a large-scale study on the correlation between genetic variation and patient survival rate in pancreatic cancer patients in Korea, and found that a mutation of a specific gene directly correlated with a patient's poor prognosis These results can be used to predict the prognosis of pancreatic cancer patients and to provide a better treatment method by strategically utilizing the above data in diagnosis and treatment of pancreatic cancer patients.

Accordingly, the present invention K- ras Gene or a nucleic acid that specifically binds to the DPC4 gene. The post-operative prognosis may be any one selected from the group consisting of metastasis, recurrence, survival and disease-free survival, but is not limited thereto.

"Nucleic acid" refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, if appropriate, ribonucleic acid (RNA). The term also encompasses both single (sense or antisense) and double helical polynucleotides, as applied to the described embodiments, as well as analogs of either RNA or DNA prepared from the nucleotide analogs (e.g., peptide nucleic acids) . In the present invention, the term 'nucleic acid' and the term 'nucleotide' are used in combination.

The term primer in the present invention refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerization reaction enzymes) Nucleotide. &Lt; / RTI &gt; The appropriate length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer of the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence of the gene that is the template, and it is sufficient that the primer has sufficient complementarity within the range capable of hybridizing with the gene sequence to perform the primer action. In addition, it is preferable that the primer according to the present invention can be used for gene amplification reaction.

The amplification reaction refers to a reaction for amplifying a nucleic acid molecule. The amplification reactions of these genes are well known in the art, and examples thereof include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR) (LCR), electron mediated amplification (TMA), nucleic acid sequencing substrate amplification (NASBA), and the like.

In the present invention, the term probe refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, which can specifically hybridize to the target nucleotide sequence, Or artificially synthesized. The probes according to the present invention may be single-stranded, preferably oligodeoxyribonucleotides. Probes of the invention can include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogs or derivatives. In addition, the probe of the present invention may also include a ribonucleotide. For example, the probes of the present invention can be used in combination with a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having C-7 substituents (the substituents being fluoro, bromo, chloro, bromo, chloro, , Iodo-, me -, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

"Subject" or "patient" in the present invention means any single entity requiring treatment, including human, cow, dog, guinea pig, rabbit, chicken, insect and the like. In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

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

Optionally, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may include compounds that are not mixed with the tissue by nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For purposes of the present invention, a "section" of a tissue sample refers to a portion or piece of tissue sample, eg, a thin slice of tissue or cell that has been cut from a tissue sample. It will be appreciated that multiple sections of a tissue sample may be taken for analysis in accordance with the present invention if the present invention is understood to include methods in which the same section of tissue sample is analyzed both at the morphological and molecular levels, It is understood that it can do.

In the present invention, "cancer "," tumor ", or "malignancy" generally indicates or describes the physiological condition of a mammal having characteristics of unregulated cell growth. In the present invention, the cancer includes, but is not limited to, pancreatic cancer, particularly pancreatic ductal cell cancer.

Further, in the present invention, the term "diagnosis" means confirming a pathological condition. For the purpose of the present invention, the prognosis diagnosis is to confirm the transformation, recurrence, survival and disease-free survival of pancreatic cancer by confirming the mutation, activation / inactivation or expression level of the post-operative gene marker in pancreatic cancer patients.

All technical terms used in the present invention in addition to the above-mentioned terms are used in the meaning as commonly understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

The present invention provides a method for diagnosing prognosis after surgery of pancreatic cancer, in addition to the above composition for prognosis after surgery for pancreatic cancer. The diagnostic method is characterized in that (A) K- ras Determining the mutation of the gene or the expression level of the DPC4 gene; And (B) predicting a prognosis after surgery for pancreatic cancer, comparing the measured value of step (A) with a reference value. The sample is preferably a tumor cell or tumor tissue, but is not limited thereto. Further, the diagnostic method K- ras When GCT, which is the 12th codon of the gene, is mutated, it is preferable, but not limited, to judge that the survival rate after pancreatic cancer surgery is low. Further, the K- ras The mutation of the gene is K- ras The GCT, which is the 12th codon of the gene, may be any mutation selected from the group consisting of GAT, GTT, CGT, TGT and CTG, but is not limited thereto. In addition, when the expression level of the DPC4 gene is decreased, it is preferable that the diagnosis method determines that the survival rate after surgery for pancreatic cancer is low, but the present invention is not limited thereto.

The gene marker according to the present invention provides information on the survival rate after diagnosis of prognosis after surgery for pancreatic cancer, that is, information on survival rate after surgery for pancreatic cancer, and information on the type, dosage, and concentration of the anticancer agent applied after pancreatic cancer surgery. That is, K- ras The GCT, which is the 12th codon of the gene, measures the mutation status and / or the expression level of the DPC4 gene to determine the kind, dose and concentration of the anticancer drug to be applied after surgery for pancreatic cancer. In particular, the kind, dose, and concentration of the anticancer drug to be loaded on the anticancer patch after pancreatic cancer surgery can be determined.

Therefore, the present invention relates to (A) a sample obtained from a pancreatic cancer patient, K- ras Determining the mutation of the gene or the expression level of the DPC4 gene; And (B) determining the type, dose, and / or concentration of the anticancer drug to be administered after the operation of the pancreatic cancer according to the measurement value of the step (A), and selecting the anticancer agent for preventing recurrence or metastasis after surgery for pancreatic cancer. Can provide a method for providing information for the user. Through the provision of the information, the present invention can provide a customized treatment method according to the genetic information of each patient, thereby improving the therapeutic effect.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

&Lt; Example 1 >

Patient group analysis

Between April 2004 and December 2008, 272 patients with pancreatic ductal cell carcinoma who underwent surgical resection at the Asan Medical Center in Seoul, Korea (mean age 60.0 years, range 22-78 years) Their medical records and pathology data were retrospectively reviewed and all experiments were conducted with the approval of the IRB.

The overall survival rate of the patients was determined by the degree of tumor including genetic mutations of four genes (K-ras, p53, c-erbB-2, DPC4), lymph node metastasis, cell differentiation, We analyzed histologic data such as embolization and clinical variables such as gender, age at diagnosis, history of preoperative diabetes mellitus, smoking, and alcohol status.

Tumor, Node and Metastasis (TNM) staging and tumor differentiation were classified and graded according to the American Joint Committee of Cancer manual 34 and the World Health Organization (WHO) nomenclature.35 .

&Lt; Example 2 >

Detection of genetic variation

Paraffin sections fixed with formalin at 5 [mu] m were obtained from all the resected tumors, and all tumors analyzed were confirmed to be primary pancreatic duct cell carcinoma.

2-1. K-ras  Mutation analysis

The K-ras tumor gene mutation was analyzed in 272 patients. Surgical specimens were classified as T3 in 93.7% (259 patients) and local lymph node metastasis was found in 57.5% (158 patients). On the other hand, 91.6% (249 patients) were diagnosed as stage IIA or IIB, and 13.6% (37 patients) showed low - grade tumors. In 89.8% (245 patients), R0 resection was performed, and immediately after surgery, there was no residual cancer by visual or microscopic examination. Peritoneal resection stones (mesenteric artery or retro pancreatitis) were microscopically examined in 27 patients (10.2% Respectively.

The mutations in the K-ras oncogene were analyzed by enriched polymerase chain reaction (PCR) -restriction fragment length polymorphism analysis, and PCR was performed using K-ras oncogene (Bp) over the codon 12 of &lt; RTI ID = 0.0 &gt; The amplification reaction using Taq polymerase was performed using 2 units of the polymerase, 50 pmol of each primer pair, 50 pmol / L of dNTP mix, 2.0-mmol / L Mg2 ⁺ , 60-mmol / L, and Tris-HCl (pH 8.8). 75 [mu] L of mineral oil was applied to the reaction mixture and amplified for 12 cycles at 96 DEG C for 1 minute, 55 DEG C for 1 minute, and 73 DEG C for 0.5 minute. Three products (114, 29, and 14 bp, respectively) were found in the non-mutant case of the PCR products using the restriction enzyme BstN1 (New England Biolabs, Beverly, Mass) , 13 bp in size).

The PCR product was subjected to a secondary PCR reaction consisting of 24 cycles in the same manner as the temperature and time conditions used in the PCR. A second PCR reaction with another primer resulted in a mutation in the first two nucleotide sequences of codon 12 when the 135 bp PCR product was cleaved into two fragments by BstN1 and not by two fragments by BstN1 It can be judged. The size of the cleaved product was determined by agarose gel electrophoresis, according to molecular weight standards.

2-2. Immunohistochemical staining (p53, c-erbB-2, DPC4)

After the deparaffinization and antigen retrieval, the slides were incubated with either p53 (clone DO-7, 1: 3000), DPC4 / Smad4 (clone EP618Y, 1: 100) monoclonal antibody or c-erbB- Lt; / RTI &gt; antibody. Monoclonal antibodies against p53 and multicellular antibodies against c-erbB-2 were purchased from DAKO (Denmark) and DPC4 / Smad4 monoclonal antibodies were purchased from Epitomics (Calif). The label was detected using the avidin-biotin complex method. (DAB) for p53 and c-erbB-2 or 3-amino-9-ethylcarbazole for DPC4 / Smad4 was used as a chromogen and physiological saline was used as a negative control Lt; / RTI &gt; for primary antibodies.

In a pathologic report on immunohistochemical staining, c-erbB-2-positive tumors were graded in the range of 0 to 3 based on the tumor cell staining intensity and intensity. Where 0 is less than 10% staining, 1 is more than 10% faint and partial membrane staining, 2 is more than 20% complete membrane staining, and 3 is more than 10% strong and complete membrane staining. In the present invention, tumors showing complete membrane staining (2 or 3) of more than 10% were considered c-erb-B-2 positive. On the other hand, the frequency of p53- and DPC4-positive cells in tumors was graded in the range of 0-3 as follows: 0; Less than 10%, 1; 10 to 33% positive, 2; 34 to 67% positive, 3; More than 67% positive. When p53 and DPC4 were less than 10% in the cytoplasm or nuclear compartment, they were considered negative.

2-3. Interpretation of genetic variation

K- ras mutation status was analyzed by PCR in 234 cases, and p53, c-erbB-2, and DPC4 gene mutations were detected by immunohistochemical staining in 272 cases.

As a result, K- ras Genetic mutations were identified in 53.8% of the patients (see Table 1). Information by PCR analysis of K- ras mutations included base transtion, nature of amino acid substitution, number of mutated codons, and reported subtype mutations include GAT ( aspartate, GTT (valine), CGT (arginine), TGT (cysteine), CTG (leucine), and AGT (serine). The wild type GGT (glycine) codon of K- ras was found in 31.2% of cases, GAT (aspartate), 14.5% of GTT (valine), 5.6% of CGT (arginine), 1.7% of TGT (Leucine) and 0.4% for AGT (serine) (see Table 2).

On the other hand, p53 or DPC4 was not expressed in 38.2% and 81.6% of patients, respectively, and c-erbB-2 was overexpressed in 7.3% of patients (see Table 1).

< Example  3>

All of the patient Survival rate

Through survival analysis, K- ras Mutation of the gene was closely related to the postoperative survival rate (p = 0.001, see Figure 1A).

Inactivation of the DPC4 gene was also found to have a significant effect on the overall survival of the patient (P = 0.047, see Figure 1D). In contrast, the genetic variation of the p53 or c-erbB-2 gene did not show a significant correlation with the overall survival rate of the patient (see FIG. 1B, 1C).

K- ras By comparing the subtype of mutation with the mean survival rate of the patient, the inventors have found that the overall survival of the patient is significantly different between the wild type and the GAT subtype (P < 0.001, see FIG. 2A). However, there was no significant difference between wild type and other subtypes and other subtypes (see Figures 2B-2D).

< Example  4>

All of the patient Survival rate  Of affecting factors Univariate  And multivariate analysis

There were eight factors influencing the overall survival rate of patients, age over 60 years, increased CA-19Y9 (937 U / ml), mutation of the k-ras gene, inactivation of the DPC4 gene, , Undifferentiated cancer, and presence of lymphangioma embolization (see Table 3).

Of the eight factors that influence the overall survival rate of the patients in the univariate analysis, six factors were statistically significant in multivariate analysis (see Table 4). (OR, 1,163), DPC4 gene inactivation (OR, 2.166), undifferentiated cancer (OR, 1.818), mutation of the K-ras gene (OR, 1631), lymph node metastasis (OR, 1.639), increased CA-19Y9 (OR, 1.328).

< Example  5>

Statistical analysis

Survival analysis and univariate analysis were performed by Kaplan-Meier method and log-rank test. P <0.05 was considered statistically significant. Multivariate analysis of significant factors identified by univariate analysis was conducted using the Cox regression model used to calculate the odds ratio with a 95% confidence interval. For statistical analysis, SPSS version 12.0K software (SPSS, Inc, Chicago, Ill) was used.

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

Claims (10)

K- ras A gene or a nucleic acid that specifically binds to the DPC4 gene. The method according to claim 1,
Wherein the post-operative prognosis is any one selected from the group consisting of metastasis, recurrence, survival and disease-free survival. The method according to claim 1,
Wherein the nucleic acid is a primer or a probe. (A) Among the samples collected from patients with pancreatic cancer, K- ras Determining the mutation of the gene or the expression level of the DPC4 gene; And
(B) predicting a prognosis after surgery for pancreatic cancer, comparing the measured value of step (A) with a reference value. 5. The method of claim 4,
Wherein the sample is a tumor cell or tumor tissue. 5. The method of claim 4,
The K- ras Wherein GCT, which is the 12th codon of the gene, is mutated, the survival rate after the operation of pancreatic cancer is considered to be low. The method according to claim 6,
Wherein the GCT is mutated into any one selected from the group consisting of GAT, GTT, CGT, TGT, and CTG. 5. The method of claim 4,
Wherein when the expression level of the DPC4 gene is decreased, the survival rate after surgery for pancreatic cancer is determined to be low. K- ras A gene or a nucleic acid that specifically binds to a DPC4 gene, and which is applied after surgery for pancreatic cancer. (A) Among the samples collected from patients with pancreatic cancer, K- ras Determining the mutation of the gene or the expression level of the DPC4 gene; And
(B) determining the type, dose, and / or concentration of the anticancer drug to be administered after the operation of the pancreatic cancer according to the measurement value of the step (A); selecting or administering an anticancer drug for preventing recurrence or metastasis after surgery for pancreatic cancer; Method of providing information for method determination.

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