KR20100034557A - Bio marker for predicting early-relapse after operation for lung adenocarcinoma - Google Patents

Abstract

PURPOSE: A biomarker for predicting initial recurrence after lung adenocarcinoma surgery by observing mutation of gene on a specific site is provided. CONSTITUTION: A biomarker for predicting initial recurrence after lung adenocarcinoma surgery contains chromosome sites 5q21.3, 7p11.2, 9q34.11, 10p11.22, 11p13, 11p15.1, 11p15.4, 12p13.31, 16p13.12, 16q22.1, 17q11.2 or 21q22.11 or nucleic acid sequence thereof. A method for predicting initial reoccurrence after lung adenocarcinoma surgery comprises: a step of extracting nucleic acids from cancer tissue or cancer cells; a step of hybridizing the nucleic acids with nucleic acids which selectively bind to the biomarker; and a step of detecting hybridization complex.

Description

Biomarker for predicting early-relapse after operation for lung adenocarcinoma}

The present invention relates to a biomarker for predicting early recurrence after lung adenocarcinoma surgery, and more particularly, in order to contribute to lowering the recurrence rate by predicting high initial recurrence after treatment of lung cancer patients, a gene of a specific region on the whole chromosome of the lung adenocarcinoma patient The present invention relates to a biomarker for identifying initial recurrence by observing mutations.

Genetic modification of cells is closely related to tumor development and progression. Tumors are known as a multi-step process involving oncogenes, tumor suppressor genes, DNA mismatch repair genes, and gene mutations associated with aging and apoptosis of cells. These include chromosomal shifts, deletions, and amplifications. May appear. Various cytogenetic or molecular genetic techniques have been used to find such genetic modifications, and the specific chromosomal abnormalities found can be applied to the diagnosis or prognosis of cancer.

Comparable genomic hybridization (CGH) provides a one-stop view of changes in relative chromosomes on all chromosomes that were not easy with conventional cytogenetic analysis. It is possible to detect the amplified or deleted part of the tumor easily according to the ratio of the binding signal of the tumor DNA to the normal DNA binding signal by labeling the DNA obtained from the tumor tissue and the normal DNA differently and then competitively binding to the normal medium chromosome. In a way that is active, there have been active studies in various solid cancers. Therefore, the CGH technique in cancer research can be useful for analyzing cancer progression, clone progression, cancer subdivision and prognosis, and to identify and analyze genes related to cancer. However, the biggest disadvantage of CGH is that the resolution is 20 Mb, which makes it difficult to detect the location of the modified gene. In addition, CGH technology must determine the chromosome induced by the medium phase on the microscope, so it is impossible to know the change of minute parts on the chromosome, so the actual resolution is more than 20Mb, and it takes a lot of time and effort due to the difficulty of manipulating and analyzing the result. Require. Therefore, a new genomic DNA chip (Array-CGH) has been devised to investigate the numerical changes of chromosomal DNA by mixing these CGH and DNA chip technologies. Array-CGH (DNA) is a DNA fragment that knows the exact location on a chromosome instead of a medium-induced chromosome on a glass slide. Its high resolution is incomparable with conventional CGH. Have The array-CGH BAC Chip can confirm the numerical change of each clone in one experiment by placing the sequence mapped BAC clone on the slide. This approach not only promotes the discovery of genes that are important factors in disease in amplicons, but can also lead to many advances in molecular diagnosis and treatment of diseases through horizontal comparisons with known gene expression information.

While various array-CGH studies have been conducted in breast cancer, there have been studies on changes in chromosome copy number according to lung cancer tissue type and changes in chromosomal changes in lung cancer cell line in lung cancer. No change studies have been made.

Lung cancer is a cancer species with high mortality rates worldwide. It is also the second most common cancer type and one of the most fatal in Korea. Lung cancer is largely divided into small cell and non-small cell cancer, non-small cell cancer accounts for about 80% of the total lung cancer, adenocarcinoma 40%, squamous cell carcinoma 40% and large cell cancer and other. Lung cancer undergoes different clinical processes for different tissue types and requires an individual therapeutic approach. Genetic modification of cells is closely related to tumor development and progression. While chromosome variation using the array-CGH method has been conducted in various array-CGH studies in breast cancer, there have been studies of chromosome copy number according to lung cancer cell type and chromosomal change in lung cancer cell line in lung cancer. The study of chromosomal copy number change with or without recurrence has not been made yet. Therefore, the present invention is to use the array-CGH method to predict the early recurrence probability by selecting the chromosomal change site having a statistical significance and clinical usefulness between the initial recurrence and non-relapse patients after adenocarcinoma of lung cancer do.

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior arts, and as a result, the difference in chromosomal changes between early relapsed lung cancer patients and non-relapsed lung cancer patients after surgery and treatment was confirmed using the array-CGH method. By selecting clones with statistical significance between groups, we confirmed that these clones could be used as predictive markers for recurrence in patients with lung adenocarcinoma, thus completing the present invention.

Therefore, the main object of the present invention is to use the array-CGH method to determine the possibility of initial recurrence by screening the chromosomal change site which has statistical significance between the early relapse patients and the non-relapse patients after surgery of adenocarcinoma patients in lung cancer. The present invention provides a biomarker for predicting initial recurrence after lung cancer surgery to be used for predicting in advance and a diagnostic kit including the same.

According to one aspect of the invention, the invention provides chromosomal regions 5q21.3, 7p11.2, 9q34.11, 10p11.22, 11p13, 11p15.1, 11p15.4, 12p13.31, 16p13. A biomarker for predicting early recurrence after lung adenocarcinoma surgery comprising one or more chromosomal sites or nucleic acid sequences thereof selected from the group consisting of 12, 16q22.1, 17q11.2 and 21q22.11 is provided.

The twelve chromosomal regions of the present invention showed that the number of DNA copies was changed in the early relapse patients of adenocarcinoma of non-small cell lung cancer compared to the non-relapsed patients (see FIG. 4).

In the present invention, the chromosome region is named according to the nomenclature of the cytoband, which is a band pattern that appears when the human medium chromosome is dyed. Specifically, the letters "p" and "q" in the chromosome region are shorter relative to the chromosome in the chromosome of the cell division in the state where two chromosomes are bound by centromere after DNA replication is completed. -arm or p-arm, the long side is called long-arm or q-arm, meaning "p-arm" and "q-arm", respectively. For example, "chromosome 8p23.1" is located in p cancer of human chromosome 8 and "chromosome 17p11.2" is located in p cancer of human chromosome 17.

To explain this further, in the late 1960s, scientists found a characteristic staining pattern on the chromosomes of quinacrine mustard stained plants (Caspersson et al., 1968). This banding technique has been applied to human chromosomes (Caspersson et al., 1971), and this band pattern can be refined and not only can normal chromosomes be easily identified without confusion, but also major structural modifications can also be characterized. It turns out that there is. Regions and bands were consecutively numbered from the mobilization outward of each chromosome arm. Specific unique band landmarks were selected to divide each cancer into regions. These landmarks belonged entirely to the end of the landmark and were considered the first band of that site. Thus, the first landmark found is the beginning of region 2. This site is divided into two or more depending on the staining pattern of the site. For example, the site 8q2 is divided into four bands to be 8q21, 8q22, 8q23, 8q24. With increased band resolution using chromosomes in the late period, some of the lower resolution bands of the medium chromosome can be further divided according to the emerging high resolution bands. If an existing band is divided into lower subdivisions, the decimal point is placed after the original band position, followed by the number that allocates the lower band. For example, gray dyed, high-resolution bands are divided into 8q22 bands and subbands 8q22.1, 8q22.2, and 8q22.3. 8q22.1 is closer to isotope than 8q22.3. If the lower band is further divided, additional numbers are added (Kuo-Ho Yen et al., Bioinformatics 2005 21 (17): 3469-3474).

"Array-CGH" used in the present invention is a DNA fragment (DNA Fragment) that knows the exact position on the chromosome instead of the medium-induced chromosome on the glass slide (Glass Slide). In the embodiment of the present invention, a BAC (Bacterial artificial chromosome) chip was used as an array-CGH, and the numerical change of each clone could be confirmed by one experiment by placing a sequence mapped BAC clone on a slide. there was.

According to another aspect of the present invention, the present invention comprises a nucleic acid sequence that can selectively bind to the biomarker, diagnostic kit for predicting the initial recurrence after lung adenocarcinoma surgery comprising a detection device for identifying the genetic variation in the biomarker To provide.

Nucleic acid sequences selectively bindable to the biomarker can be obtained from the cloned sequence-mapped to the array-CGH. For example, referring to Table 1, as in the case of Airy-CGH used in the present invention, the mapping position of cytoband 11p15.4 in BAC clone 202 is 2811177-2898536 and some or all of the sequence mapped BAC clones May be a nucleic acid sequence capable of binding to the biomarker of the present invention.

In the diagnostic kit of the present invention, the detection apparatus may be a fluorescence microscope for detecting the FISH analysis result or an image scanner for detecting the microarray analysis result. Will be measured. The DNA obtained from the tumor tissue and the normal DNA are differently labeled and then competitively combined with the biomarker to detect amplification or loss in the tumor according to the ratio of the binding signal of the normal DNA to the tumor DNA binding signal. Genetic variation is observed when the ratio of the combined signal is about 0.25 or more and -0.25 or less.

In an embodiment of the present invention, the gDNA of the tumor tissue is labeled as Cy3, the reference gDNA is labeled as Cy5, and the log value of the Cy3: Cy5 binding signal ratio using an image scanner (detector) after binding with the biomarker. It was determined that the gain of 0.25 or more was gain and the loss of -0.25 or less.

In the present invention, the diagnostic kit may be a DNA chip.

As a method for preparing the DNA chip, a conventional method may be used as shown in Table 1 below.

TABLE 1 Conventional DNA Chip Manufacturing Method

More specific DNA chip manufacturing method is generally as follows. Using the DNA sequences that have been identified, the primers, which are necessary for PCR (Polymerase Chain Reaction) at the beginning and end of these DNA sequences, are found by computer. These primers have similar annealing temperatures, so they must be able to PCR multiple DNA sequences at the same time. After PCR, success is examined on an agarose gel and the amplified DNA sequences are concentrated to an appropriate concentration. The amplified and concentrated DNA sequences are then planted on glass slides, which are often used in microscopic experiments with a microarray machine. In addition to the poly-L-lysine glass, the glass may be a glass coated with silane, silylate, or super-amine. Coating of the glass is necessary for fixing the probe carried by the microarray. In order to fix the probe, a base capable of complementarily covalent bonding with a material coated on the glass may be inserted at a 3 'or 5' position of the probe. For example, in order to fix the probe to the glass coated with the amine group, an aldehyde group is inserted using a linker column at the 3 'or 5' position of the probe. In addition, in order to facilitate a covalent bond reaction, the reaction is induced at an appropriate humidity for 1 hour or more, and left for 6 hours or more to fix the DNA probe.

In the analysis of the DNA chip, it is possible to determine whether the genetic mutation by labeling the sample nucleic acid with Cy3 and Cy5 in the same manner as mentioned above and then bound to the DNA chip to measure the binding strength.

According to another aspect of the present invention, the present invention comprises the steps of extracting a nucleic acid from cancer tissue or cancer cells of a lung cancer patient; Hybridizing the nucleic acid with a nucleic acid sequence capable of selectively binding to the biomarker of claim 1 to form a hybrid complex; And it provides a method for predicting the initial recurrence after lung adenocarcinoma comprising the step of detecting the hybrid complex.

In the prediction method of the present invention, a fluorescent material such as Cy3-dUTP and Cy5-dUTP may be used to detect the hybrid complex. These fluorescent labels are for measuring the intensity of genetic variation due to any color difference. When measuring the intensity of the mutation, Cy3 becomes green at 532nm and Cy5 becomes red at 635nm. In addition to the fluorescent materials exemplified above, various labeling materials may be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these examples.

Test subject

The cancer tissues were collected from 21 cases of adenocarcinoma diagnosed with lung cancer and surgical or tissue biopsy at Korea University Medical Center from November 2001 to October 2004. The patient's frozen lung tissue specimens were provided by a nationally designated frozen lung tissue bank and performed with the approval of the Institutional Review Board (IRB) of the ancient medical center. Early-relapse patients were relapsed within 2 years after surgery and 11 cases were non-relapsed. Non-relapse patients were 10 cases without relapse within 2 years. The average age of non-relapsed patients was 62 years old, 5 males and 5 females. The average age of early relapsed patients was 64 years old, 5 males and 6 females.

Example 1 Extraction of Tissue Genomic DNA

Extraction of genomic DNA from each tissue obtained in the test subject was performed using a PUREGENE DNA purification kit (Gentra, Minneapolis, MN). After homogenizing 600 μl of lysis solution (lysis solution) in 10-20 mg of frozen lung tissue, 3 μl of proteinase K solution was added and mixed, followed by reaction for 6 hours in a 55 ° C. thermostat. 3 μl of RNase A was added and then placed at 37 ° C. for 1 hour. 200 μl of protein precipitation solution was added and centrifuged at 13,000 rpm for 5 minutes, 600 μl of isopropanol was added to the supernatant, gently mixed, and centrifuged at 13,000 rpm for 5 minutes. The supernatant was discarded, and 600 µl of 70% ethanol was added to the DNA precipitate, followed by centrifugation for 2 minutes. The supernatant was discarded and dried at room temperature for 5 minutes. 100 μl of DNA hydration solution was added and placed at 65 ° C. for 1 hour.

Example 2. Array-Comparative Genomic Hybridization (array-CGH) Analysis

The MACArray ™ -Karyo 1.4K BAC-chip (Macrogen, Seoul, Korea) consists of 1440 bacterial artificial chromosome (BAC) clones, the microarray template whose location was determined by FISH, with resolutions up to 2 Mbp. Information on the sequence and chromosomal region of each clone number can be found at www.macrogen.com/kor/biochip/genelist.jsp.

Cy3 was labeled on gDNA of tumor tissue and Cy5 on control gDNA using Invitrogen Random priming labeling Kit (Invitrogen, Carlsbad, Calif.). The method is as follows. Add 0.5 µg of distilled water to 0.5 µg of purified lung adenocarcinoma patients and 0.5 µg of control gDNA (Promega, Madison, WI), and add 20 µl of random reaction solution (Invitrogen, Carlsbad, Calif.). After denaturation for 5 minutes, it was placed on ice for 5 minutes. Each denatured DNA was labeled with 3 μl of Cy3 and Cy5, mixed with 1 μl of klenow fragment and incubated at 37 ° C. for at least 16 hours. Each labeled DNA was purified using a Qiagene spin column (Qiagene, Valencia, Calif.) And then hybridized to a MACArray ™ -Karyo 1.4K BAC-chip (Macrogen, Seoul, Korea). The reaction was carried out for 72 hours in a hybridization chamber (hybridization chamber) at 37 ℃.

After completion of the hybridization reaction, it is washed with a washing solution (50% formamide, 2X SSC) and dried, followed by Cy3: Cy5 intensity using ArrayScanncer ™ (Macrogen, Seoul, Korea) and MAC Viewer v1.6.3 (Macrogen, Seoul, Korea). The log value of ratio was obtained and gain (0.25 or more) and loss (-0.25 or less) were analyzed.

As a result of the analysis, the gene location of the clone was estimated using the University of Calfonia, Santa Cruz (UCSC) human genome database (http://www.genome.ucsc.edu). The estimation results are shown in Table 2 below.

1 is a result of analyzing the frequency of gain (loss) or loss of the whole chromosome of adenocarcinoma patients (AdCC) using the array-CGH, Figure 2a and 2b is the initial non-recurring patients (Non) -relapse) and early-relapse of the entire chromosome acquisition or loss frequency analysis results.

Example 3 Statistical Significance Analysis of Candidate Clones

Candidate clones showing statistically significant differences between patient groups (initial relapsed patients and non-relapsed patients) were selected through a false discovery rate (FDR) test based on the frequency results for each group of Example 2, and their cytology The band (cytoband) was confirmed. Here, the FDR test solves the problem of multiple testing because the random error increases as the number of tests (N) increases in the case-control study (eg 100K, 1M chip, expression chip). It is one of the analysis methods developed for the purpose, and it is a very useful method for the association study using a large number of genetic markers. In this embodiment, as listed in R 2.6.0 software (http://r-project.org) was performed using the FDR test p order starting with the largest values of the p -value and a -value of 12 or less 0.05 Candidate clones were selected as shown in Table 2 below.

TABLE 2 Candidate clones with statistical differences between the initial and non-recurring groups

BAC_NO; BAC clone ID

BAC_start; Start position of BAC clone in cytoband

BAC_end; End position of BAC clone in cytoband

Cyto .; Cytobands, Corresponding Chromosome Locations in Clones

Genes; Genes present at locations identified through the UCSC site ( http://www.genome.ucsc.edu )

p -value; Statistical analysis of the difference between the initial and non-recurring groups through FDR test

N-gain (n); The number of gains identified by the gain of array CGH in the non-relpase group (n)

N-loss (n); (N) Number of confirmed loss in array CGH analysis in non-relpase group (n)

E-gain (n); Number of gains identified by array CGH analysis in the early-relpase group (n)

E-loss (n); Number of confirmed loss in array CGH analysis in early-relpase group (n)

In Table 2, the BAC clone IDs 202, 839, 1156, 1245, 1504, 469, and 810 in the N-group (non-recurring) were analyzed by gain, and the BAC clone IDs 1406, 1112, 1250, 1578, and 574 were lost. Analyzed. In other words, BAC clone ID 202 (chromosomes 2811177 ~ 2898536) gained 8 out of 10 patients (80%) with a log ₂ ratio of 0.25 or more, and 3 out of 11 patients with initial relapse (27.3%). ) Was determined as gain. The BAC clone ID 1406 (Chromosome No. 106757798 ~ 106856173) was found to have a log ₂ ratio of less than -0.25 in 5 non-relapsed patients (50%) and a change in the copy number of the site in the initial relapse group. There was no. BAC clone ID 839 (chromosome 55002043 ~ 55102035) was found to be gained only in 4 non-relapsed patients (40%), and BAC clone ID 1112 (chromosome 5580555 ~ 5683898) was identified in 3 patients (30). %), BAC clone ID 1156 (chromosome 16 13369407-13465245) gained only in 4 non-relapsed patients (40%), and BAC clone ID 1245 (chromosome 9 132266880-132364697) was non-recurring 3 Gain in 30%, BAC Clone ID 1250 (Chromosome Nos. 32657344-32743665) lost in 4 non-recurring groups (40%), BAC Clone ID 1504 (Chromosome No. 34544563-34630524) in four non-recurring groups ( 40%) gain, BAC clone ID 1578 (chromosome 17246684 ~ 27409341) was lost to 5 non-recurred groups (50%), and BAC clone ID 469 (chromosome 16 to 67959410-68054859) was non-recurred 3 patients (30%). ), Loss of BAC clone ID 574 (chromosome 21 33763179 ~ 33831886) in 4 non-relapsed patients (40%), loss of BAC clone ID 810 (chromosome 11 19817846-19982397) in non-relapsed 4 patients (40%). with gain It was determined. As described above, the chromosomal parts showing different changes between the non-recurring patient group and the initial recurring patient group were screened by the array CGH method, and 12 sites of chromosomal variation that were specifically different from the statistically significant non-recurring group were identified. Therefore, by analyzing the gain or loss of the chromosomal region corresponding to the clone from the lung adenocarcinoma surgery patients can be predicted after the initial recurrence, each of these can be used as a marker for predicting the initial recurrence after the lung adenocarcinoma surgery. In addition, the accuracy of the initial recurrence prediction may be further improved by analyzing the combination of the markers.

In FIG. 3, a clone selected by performing hierarchical clustering using Cluster 2.11 software (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm). Their significance was again confirmed. Instrumental clustering analysis of the 12 clones selected showed that the N-groups (non-recurrences) and the E-groups (initial relapses) were similarly divided, although not completely divided into two clusters between the two groups.

In Table 3 below, sensitivity and specificity were calculated according to the cutoff value, which is the number of clones changed among the 12 clones shown in Table 2, and when the cutoff value was 4, the most significant sensitivity and It was confirmed to have specificity.

TABLE 3 Sensitivity and Specificity of Cutoff Values in 12 Significant Clones

In addition, in FIGS. 5A and 5B, the relapse free survival (survival without recurrence after surgery and treatment) was compared with the cutoff value of selected clones using the Kaplan-Meier method, and the ROC curve (Receiver operating characteristic curve) Was obtained and its significance was confirmed. SPSS 10.0 software (SPSS Inc., Cicago.IL) was used for the analysis. Specifically, when the cutoff value is 4 in FIG. 5A, the area under the curve (AUC) is 0.894 and the p- value is 0.002, and a statistically significant value is obtained, and when the cutoff value is 4 in FIG. Statistically significant ( p -value 0.00016) relapse period was found to be long.

Example 4 Fluorescence In situ Hybridization (FISH)

Paraffin tissue made slides washed and dried. The probe was MacProbe (Macrogen, Seoul, Korea) fabricated in the same size for the same site as the BAC clone used in 1.4 BAC-chip (Macrogen, Seoul, Korea). This allows us to reconfirm exactly the same clones identified in the array CGH results. After denaturing at 75 ° C for 5 minutes using 10 μl dual hybridization mix containing probes labeled with fluorescein isothiocyanate (FITC) and rhodamine, respectively, and then denatured at 75 ° C for 5 minutes. The reaction was allowed to react for 16 hours at. Probe labeling was MacProbe solution (Macrogen, Seoul, Korea). After washing the dried slides were confirmed by fluorescence microscope (Leica DM RXA2) fluorescence signal to determine the gain (loss) or loss (loss) of the clone. The ratio of the red / green fluorescent label was counted within 100 cells of the observed tissue, and the ratio value was determined by the t- test between the target group and the control group.

In FIG. 4, the gain and loss of the clones were reconfirmed using the FISH method for six clones among the 12 clones selected in Table 2, which are sites for FISH analysis, and array-GCH results and A match was confirmed.

4, the Rhodamin (orange) label is a test clone selected from an array CGH analysis, and the FITC (green) label corresponds to a control group with a BAC clone at a position that does not show a change in the same chromosome. The left panel confirms that Rhodamin (orange) and FITC (green) labels are labeled one copy in normal cells and the right panel is the result in tumor tissue. FIG. 4A is a result of labeling BAC no.839-Rhodamine at 7p11.2 and control BAC no.1418-FITC and confirming that the test clones (orange markers) were more gained (arrows) on non-recurrent tissue slides. . 4B was identified as the loss of the control clone (green marker) as the 12p13.31 position was marked by # 1112-Rhodamine, # 766-FITC on the non-recurrent tissue slide. 4C was identified by labeling 16p13.12 with # 1156-Rhodamine, # 357-FITC on non-recurrent tissue slides, and the test clone (orange marker) was found to have more gain. 4D was identified by labeling 9q34.11 as # 1245-Rhodamine, # 681-FITC on non-recurring tissue slides, and the test clone (orange marker) was found to have more gain. 4E identified the 11p13 position as # 1504-Rhodamine, # 934-FITC on non-recurrent tissue slides, and the test clone (orange marker) was found to have more gain. 4F was identified as loss by labeling 21q22.1 with # 574-Rhodamine and # 654-FITC on non-recurrent tissue slides. This result is the same as that of the clone selected with the FDR test from the array CGH analysis.

Example 5. Confirmation of DNA copy number change through real-time qPCR

Primers used in the experiment was prepared in consideration of the gDNA sequence of candidate genes confirmed by the array-CGH analysis. 10 μl of 2X IQ ™ SYBR green supermix (Bio-Rad, Hercules, CA), 1 μl of DNA, 1 μl of each primer (10 pmol / μl) in total 20 μl reaction solution Real-time qPCR was performed. GAPDH gene and the human whole blood male (male human whole blood) and the DNA used as a control 2- ^ΔΔ _t C method (Whang G et al, Nucleic Acids Res 2004; 32 (9):.. E76, Livak KJ and Schmittgen TD Methods Relative DNA copies were calculated as 2001; 25 (4): 402-8).

Primer sequences used in real-time qPCR are shown in Table 4 below.

[Table 4]

In Table 5 below, DNA copy number changes of genes related to cancer development, progression or metastasis among the genes present in the clone region confirmed by array-CGH results by real time qPCR were confirmed. DNA clones were performed by real-time qPCR of 11 genes included in the chromosomal clone site among candidate genes included in the chromosomal clone site among 11 clones except one clone of 12 selected clones. As a result of confirming the numbers, except for the ASS and TMEM16B genes, it was confirmed that almost identical to the array-CGH results.

TABLE 5 Real-time qPCR method using genomic DNA confirms DNA copy number changes of candidate genes

gain n (%) / loss n (%); The number of samples, n (percentage%), determined as the gain or loss of the analysis by each analysis method.

In the above results, the 12 clones identified in adenocarcinoma, in particular, have been identified as useful markers to distinguish between early and non-recurrence. Through the present invention, useful markers for predicting the early and non-recurrence of adenocarcinoma among non-small cell lung cancers were identified using the array CGH method, and candidate genes present in the site of chromosome change through FISH analysis and real-time qPCR. It was confirmed that this is related to the change in copy number. Based on these results, we expect the clinical usefulness to predict the early recurrence after surgery by using the marker of specific chromosomal variation in adenocarcinoma.

As described above, according to the present invention, the difference in chromosomal changes between patients with early recurrent lung adenocarcinoma and patients with non-recurring lung adenocarcinoma using the array-CGH method, and by selecting clones having statistical significance between the two groups, Chromosomal changes can be selected and used as biomarkers to predict early recurrence after export or treatment in patients with lung adenocarcinoma.

1 is a result obtained by analyzing the frequency of the gain (loss) or loss (loss) of the entire chromosome of adenocarcinoma patients using the array-CGH through the array scanner device.

Figure 2a is a result obtained by analyzing the frequency of acquisition or loss of the whole chromosome of the initial relapse patients using the array-CGH through the array scanner device.

Figure 2b is a result obtained by analyzing the frequency of acquisition or loss of the entire chromosome of non-recurring patients using the array-CGH through the array scanner device.

3 shows Unsupervised hierarchical clustering analysis of 12 clones selected from adenocarcinoma patients.

4 is a result of re-identification of the clones confirmed the array-CGH results by the FISH method.

Wherein Rhodamin-labeled target (orange), FITC-labeled control (green) BAC clones. A, # 839-Rhodamine, # 1418-FITC. B, # 1112-Rhodamine, # 766-FITC. C, # 1156-Rhodamine, # 357-FITC. D, # 1245-Rhodamine, # 681-FITC. E, # 1504-Rhodamine, # 934-FITC. F, # 574-Rhodamine, # 654-FITC.

5A and 5B show ROC curve analysis and Kaplan-Meier survival curves for cutoff values according to the changed number of clones of 12 clones, respectively.

Claims (3)

Chromosomal regions 5q21.3, 7p11.2, 9q34.11, 10p11.22, 11p13, 11p15.1, 11p15.4, 12p13.31, 16p13.12, 16q22.1, 17q11.2, and 21q22 in the human genome . Biomarkers for predicting early recurrence after lung adenocarcinoma surgery comprising one or more chromosomal sites selected from the group consisting of .11 or nucleic acid sequences thereof. It comprises a nucleic acid sequence selectively bindable to the biomarker of claim 1, Diagnostic kit for predicting early recurrence after lung adenocarcinoma surgery comprising a detection device for identifying the genetic variation in the biomarker. Extracting nucleic acids from cancer tissue or cancer cells of a lung adenocarcinoma patient; Hybridizing the nucleic acid with a nucleic acid sequence capable of selectively binding to the biomarker of claim 1 to form a hybrid complex; And Early recurrence prediction method after lung cancer surgery comprising the step of detecting the hybrid complex.

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