KR100945917B1 - Prediction of drug-responsibility against breast cancer through chromosomal aberration detection - Google Patents

Abstract

PURPOSE: A method for predicting drug reactivity of breast cancer patient is provided to measure increase and decrease of duplication number of specific chromosome site and quickly and accurately predict drug reactivity. CONSTITUTION: A composition for predicting reactivity of drug such as docetaxel or doxorubicine in breast cancer patient contains 20-100bp of oligonucleotide positioned in genome DNA fragment 17q21.32-33, 100bp-100kbp cDNA derived the genome DNA fragment, genome DNA fragment, olidonucleotide and cDNA complementary sequence. The prediction of drug reactivity for a breast cancer patient is performed by comparing the number of duplication of 17q21.32-33 site of human chromosome 17 in a DNA sample obtained from the breast cancer patient. The comparing the number of duplication is performed through PCR, array-based analysis, or fluorimetry.

Description

Prediction of drug-responsibility against breast cancer through chromosomal aberration detection}

Use as a marker for predicting drug reactivity of breast cancer patients in a certain region of human chromosome 17, composition for predicting drug reactivity in breast cancer patients comprising the same, and whether the specific region of the human chromosome 17 is missing or amplified Methods of predicting drug responsiveness in breast cancer patients are provided.

In locally advanced breast cancer, only 40-60% of patients undergoing chemotherapy prior to surgery have been reported to respond. Such chemotherapy is performed to reduce the size of the tumor before surgery, but the problem is that the size of the chemotherapy is often not reduced. That is, although some patients suffer from unnecessary side effects, most patients receive chemotherapy. Side effects of drug-based anticancer therapy include digestive side effects such as nausea, vomiting, loss of appetite, diarrhea, constipation, abdominal pain and fever due to leukocytes, red blood cells, thrombocytopenia, decreased bone marrow function such as anemia, and secondary infections due to decreased immune function. , Hair loss, and reproductive harm. In addition to these serious biological side effects, considering the cost of chemotherapy, it seems unreasonable to preoperative chemotherapy for ineffective patients. However, there are no known markers for predicting the response of chemotherapy, and there are cases in which the effect of chemotherapy is significantly reduced or completely eliminated before surgery. It is the situation.

Recently, academia has reported that genomics research has resulted in the modification of the genome in cancer cells, which affects the expression of genes, resulting in differences in drug reactivity among individuals. Therefore, the core of this patent is to improve the quality of life by developing markers on the genome that can predict the drug reactivity of breast cancer by individual, reducing unnecessary financial cost of patients and avoiding side effects of chemotherapy. This study observes the variation of the whole genome of breast cancer patients to find out what contributes to the determination of drug responsiveness of the actual breast cancer, and proposes a model to determine the breast cancer drug reactivity.

Accordingly, the present inventors have completed the present invention by finding a specific chromosome region in which the copy number is increased or decreased depending on drug reactivity.

Thus, one example of the present invention provides a novel use as a marker for predicting breast cancer drug reactivity at specific chromosomal sites.

Another example of the present invention provides a composition for predicting breast cancer drug reactivity comprising the specific chromosome region.

Another example of the present invention provides a method for predicting breast cancer drug responsiveness by investigating copy number increase and decrease of the specific chromosome region.

The present invention is directed to a novel use as a marker for predicting breast cancer drug reactivity at specific chromosomal sites.

That is, the present invention is to select a specific genomic region that shows a difference in the number of copies in the group that responds to the drug treatment and the group that does not respond to the drug treatment among breast cancer patients, and use as a marker for predicting the drug reactivity of the selected specific chromosomal region of breast cancer patients To provide.

Genomic sites selected as markers for predicting drug reactivity of such breast cancer patients are shown in Table 1 below.

TABLE 1

number Position in the human genome Size (bp) chromosome Cytoband start End One 17 17p12 11,200,001 15,900,000 4.7 Mbp 2 17 17q21.32-33 41,900,001 47,600,000 5.7 Mbp

(According to UCSC Genome Browser on Human Mar. 2006 Assembly)

Therefore, in one embodiment, the marker for predicting breast cancer drug reactivity provided by the present invention comprises at least one genomic DNA fragment selected from the group consisting of 1 and 2 in Table 1; And it may be one or more selected from the group consisting of 20 bp or more, preferably 20 to 100 bp oligonucleotides located continuously in the genomic DNA fragment.

Drugs capable of predicting reactivity according to the present invention may be one or more selected from the group consisting of docetaxel and doxorubicin.

In addition, the marker can be used as a probe for detecting a corresponding portion of the sample.

Thus, another example of the present invention includes at least one genomic DNA fragment selected from the group consisting of 1 and 2 in Table 1 above; Oligonucleotides of at least 20 bp, preferably 20 to 100 bp, located consecutively within the genomic DNA fragment; CDNA of at least 100 bp, preferably 100 bp to 100 kbp derived from said genomic DNA fragment; And one or more probes selected from the group consisting of the genomic DNA fragments, oligonucleotides, and the complement of cDNA.

In addition, the probe is not particularly limited, but the genomic DNA fragments may include Bacterial Artificial Chromosome (BAC), Human Artificial Chromosome (HAC), Transformation competent Artificial Chromosome (TAC), Yeast Artificial Chromosome (YAC), P1-derived Artificial Chromosome (PAC). , P1 (phage), may be prepared by inserting into a vector selected from the group consisting of plasmid and cosmid (cosmid), preferably may be prepared using a BAC (Bacterial Artificial Chromosome) vector.

In another aspect, the present invention compares the copy number of a 17p12 site, 17q21.32-33 site, or both of human chromosome 17 in a DNA sample obtained from a breast cancer patient with the copy number of that chromosome site in a normal sample. It provides a method for predicting drug reactivity of a breast cancer patient comprising the steps of. The method reduces the number of copies of the 17p12 region of chromosome 17 in the DNA sample obtained from breast cancer patients compared to the number of copies of the chromosomal region of the normal DNA sample, or the 17p21.3 site of chromosome 17 in the DNA sample obtained from breast cancer patients. If the number of copies of the increased relative to the number of copies of the chromosomal region of the normal DNA sample, characterized in that the patient is determined to be non-reactive breast cancer drug.

In the present specification, the normal DNA sample may mean a DNA sample of normal cells obtained from a person who does not have a history of breast cancer, symptoms, and signs. The DNA sample obtained from the breast cancer patient may be obtained from breast cancer cells of the breast cancer patient.

DNA copy number aberrations (DNANAs) are one or more methods selected from the group consisting of all DNA quantification methods commonly available, such as polymerase chain reaction (PCR), array-based assays, fluorescence assays, and the like. Although it can measure, it is not limited to this.

PCR is the most commonly used DNA quantification method, for example, can be performed by real time (PCR), semi-quantitative PCR and the like.

Array-based assays measure the degree of hybridization using an array on which a probe capable of hybridizing with a target chromosome site is immobilized to measure the increase or decrease of the number of copies of the target chromosome site with respect to the entire genome. For example, array-based comparative genome hybridization ( cDNA, Oligonucleotide or BAC clone Array-based comparative genomic hybridization (aCGH) assays. The aCGH assay, one of the comparative genomic hybridization (CGH) methods, has superior resolution compared to the conventional CGH method and enables high resolution locus-by-locus measurement by performing one or more copies of DNA. It is a way. Because malignant tumors, including breast cancer, are accompanied by many nonspecific gene abnormalities, it is not easy to identify genetic abnormalities associated with biological properties such as reactivity to certain drugs. In the present invention, the aCGH assay is used to demonstrate the association between genomic loci with clone count abnormalities and the biological properties of tumors to detect numerical abnormalities of genomes associated with biological properties such as drug reactivity that were not easily identified. The identification was made and the basis for determining the biological characteristics and histological type of the tumor was established. To date, no technique has been reported for distinguishing reactivity of breast cancer drugs using aCGH data analysis.

CGH is a widely used technique for analyzing the whole genome and measuring amplification and deletion of specific sites on a chromosome. Recently, aCGH is a technology that combines DNA microarray technology to enable large-scale analysis on a chip. aCGH can be used to measure chromosomal aberrations in samples, and this approach not only facilitates the discovery of genes that are important factors in the disease within amplicons, but also compares the known molecular expression data to molecular diagnosis and It can bring a lot of progress to treatment. By aCGH analysis, changes in chromosomal abnormalities can be directly expressed by using an analysis program, and it is easy to distinguish between genes with increased and reduced copies.

In aCGH analysis, it may be determined whether the number of DNA copies is abnormal based on the log 2 ratio of the number of DNA copies. In the present specification, the 'log2 ratio of DNA copies' is the log2 ratio of DNA copies of the test sample to the DNA copies of the normal sample at each chromosome site (the number of DNA copies of the test sample / the number of DNA copies of the normal sample). ) Is converted to a log2 value, and more specifically, the following definition: The log2 value of the DNA copy number

= log2 (fluorescence signal in test sample * / fluorescence signal in normal sample **)

In the above formula,

* Is the signal level of the fluorescent material used to detect the number of DNA copies in the test sample, the fluorescent material used may be any fluorescent material available for DNA detection (e.g. Cy3, etc.) Measured by a method commonly used for signal detection of fluorescence material;

** is the signal level of the fluorescent material used to detect the number of DNA copies in a normal sample, the fluorescent material used may be different from that used in the test sample among all the fluorescent materials available for DNA detection ( For example, Cy5, etc.), the signal degree is a value measured by a method commonly used for signal detection of the fluorescent material used.

In an embodiment of the present invention, when the 'log2 ratio of DNA copy number' is greater than 0, preferably 1 or more, more preferably 1.25 or more, the number of copies of the corresponding genomic region is reported to be increased, and 0 or less, preferably- If it is 1 or less, more preferably -1.25 or less, it can be seen that the number of copies of the genomic region is reduced. However, the limit value for determining the chromosome abnormality may vary depending on the technique or apparatus used, and those skilled in the art to which the present invention belongs, the threshold value to the technique or apparatus used Can be determined accordingly.

The fluorescence assay measures fluorescence intensity generated by hybridizing a probe capable of hybridizing to a target chromosome site and hybridizing it to a chromosome of a test sample. For example, a FISH (Fluorescent in situ hybridization) method may be used. In an embodiment of the present invention, a probe capable of detecting a water droplet abnormality of the 17p12 region or the 17q21.32-33 region of human chromosome 17 is formed and labeled with a fluorescent substance, and then hybridized to the chromosome of the test sample. The abnormality of the target part of a probe can be discriminated.

According to the method for predicting drug reactivity of a breast cancer patient of the present invention, the number of copies of the 17p12 region and / or 17q21.32-33 region of human chromosome 17 is abnormal, that is, the number of copies of the 17p12 region is reduced, and / or 17q21.32- Increasing the number of copies at 33 sites has been detected, and breast cancer patients who have been shown to be drug-responsive to docetaxel and / or doxorubicin can avoid unnecessary drug treatment prior to surgical operations, thereby alleviating physical, mental and economic pain. In addition, it can greatly contribute to improving the quality of life during the treatment of breast cancer patients.

Breast cancer drug reactivity prediction technology by measuring the copy number increase and decrease of specific chromosome sites provided in the present invention to accurately and accurately predict the drug reactivity of individual breast cancer patients, thereby avoiding the physical, mental and economic pain of patients due to unnecessary drug treatment It can be alleviated, which is very meaningful given the quality of life during the treatment period of the patient.

Hereinafter, the present invention will be described in more detail. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited to these examples.

Example  One: ArrayCGH  base Marker  Selection

1.1. ArrayCGH  Primary Gene Sample Collection for Experiments

Sixty-six samples of breast cancer patients were obtained from Seoul National University's Breast Cancer Center. Among them, 63 patients were treated with anti-cancer drugs (docetaxel and doxorubicin). Individual drug reactivity was determined by comparing volume sizes extracted from MRI images of tumor masses before and after drug administration of 63 patients treated with docetaxel and doxorubicin.

1.2. ArrayCGH  Chip Platform

Using DNA array of Macrogen's MacArray ™ Karyo 4000 chip, the DNA dosage of the whole genome was measured at 1Mb resolution.

1.3. ArrayCGH  Experiment

By using the micro-dissection method as shown in Figure 1, cancer tissue samples were taken except normal tissues. Genomic DNA was extracted from the collected tissue samples, and ArrayCGH experiments were performed according to Macrogen's standard experimental protocol (Hwang et al. "Genomic copy number alterations as predictive markers of systemic recurrence in breast cancer" Int. J.) Cancer: 123, 1807-1815 (2008))

1.4. ArrayCGH  Drug reactivity relevance testing of the entire genome Marker  Selection

Each sample was scanned with Genepix and the images were analyzed with MacViewer ™ software. At this time, except for the case where the intensity is low or there is a problem in the spot, the average (median value) of the normalized log ₂ intensity ratio from which the noise is removed was determined to determine the dose of each DNA fragment. For each probe (clone), log ₂ (signal strength of the test sample / signal strength of the reference sample) value uses the active threshold, that is, the smoothing function, and the actual chip-to-chip variation ( The copy number aberration of the DNA of gain / normal / loss was measured in each sample by classifying according to the variation in consideration. After determining the number of copies of the entire genome of each individual, a statistical test was performed to determine whether the reactivity of the anticancer therapy was significantly different according to the abnormality of the corresponding genomic region for each probe.

To quantify drug reactivity for each individual, we used Reduced Volume Ratio, a criteria based on the volume of tumors extracted from pre- and post-dose MRI images, as follows: Reduced Volume Ratio = (Volume of pre-administration-tumor after dosing) Volume) / (volume of tumor before administration).

For the genomic region corresponding to each probe, after dividing the patients into normal groups (when chromosome duplications were 2) and abnormal groups (when chromosome duplications decreased or increased), there was a significant difference in the mean of the reduced volume ratio between the groups. A standard t-test was performed to determine whether or not For example, the upper figure (Region A) of Figure 2 shows that if a breast cancer patient has a defect in a specific genomic region, drug reactivity is significantly lower than that of a group with normal copy number, and the lower figure (Region B) In the case of amplification at the site, the drug reactivity may be increased compared to the normal group.

Using the t-test as described above, candidate probes (clone) showing a significant correlation with drug reactivity are summarized in Table 2 below. (p value <0.05)

TABLE 2

Based on previous genomic studies on the function and drug responsiveness of the genes included in the above 24 clones, two sites, 17p12 and 17q21.32-33 of chromosome 17, were selected from the Seoul National University Breast Cancer Center. In other words, after examining the functionalities of the genes included in each chromosome region with breast surgeons at the Seoul National University Breast Cancer Center, they appear to be related to the prognosis of breast cancer, that is, the regions that appear to be true positive, 17p12 and 17q21.32 Two sites of -33 were selected. For example, in the case of chromosome 8, it is difficult to exclude false positives that are frequently observed in other breast cancer chromosomes without association with drug reactivity.

In the data obtained in this example, the genome of the patients showing the deletion of the 17p12 region is shown in FIG. 3. The X axis of the figure represents the position of the entire chromosome and the Y axis is log ₂ (signal strength of the test sample / signal strength of the standard sample). In each graph, a blue vertical line indicates a 17p12 region.

In addition, the genome of the patients showing amplification of the 17q21.32-33 region is shown in FIG. The blue vertical line in each graph represents the 17q21.32-33 area.

Independent patient groups were validated to confirm the predictive power of the drug reactivity before breast cancer surgery of the two marker-based models discovered using ArrayCGH.

Example  2. FISH based Marker  Marker Validation

In addition to obtaining new patient samples from Seoul National University Breast Cancer Center, in order to confirm that the results obtained in the present invention are independent of analytical techniques, fluorescence in situ hybridization (FISH) is a method for detecting chromosomal abnormalities commonly used in pathology, not ArrayCGH. Was used to confirm the marker selected in Example 1 above.

2.1. Sample collection for use in FISH validation

A total of 21 breast cancer patients (breast cancer cells) were obtained from the Breast Cancer Center of Seoul National University Hospital.

2.2. FISH Probe

FISH probes for measuring abnormalities of the aforementioned 17p12 and 17q21.32-33 sites in the 21 samples were prepared as shown in Table 3 below. This probe was obtained using Macrogen Co., Ltd. BAC library, and a probe was prepared by attaching two consecutive clones for signal brightness.

[Table 3]

BAC clone reference information used to make FISH probe

Plate_No Chromo_No BAC_START BAC_END Target1 2559 17 45890122 46024732 2063 17 45965673 46071397 Target2 2934 17 12773307 12867433 2226 17 12800597 12955283

TABLE 4

Final FISH Probe Information

Chromo_No BAC_START BAC_END 17q21.32-33 17 45890122 46071397 17p12 17 12773307 12955283

2.3. FISH experiment

FISH experiment was performed according to standard protocol at Seoul National University Hospital Pathology (Park et al. "EGFR gene copy number in adenocarcinoma of the lung by FISH analysis: investigation of significantly related factors on CT, FDG-PET, and histopathology." Lung Cancer 64 (2): 179-186 (2009)).

2.3. FISH based Clones  decision

As shown in FIG. 5, the results showing the 17p12 deletion among the results obtained by performing the FISH as described above. Overall, the red signal representing the target 17p12 site in the sample (cancer cell) is hardly observed compared to the green signal representing the centromere of chromosome 17. Conversely, the results showing amplification of 17q21.32-33 among the results obtained by performing FISH are shown in FIG. 6. As shown in FIG. 6, it can be seen that the red signal representing the target 17q21.32-33 site is significantly higher than the green signal representing the chromosome 17.

When the amplification / deletion is not clearly distinguished as described above, the red signal and the green signal was counted for each cell. Basically, the gene copy number status was determined according to the EGFR FISH standard of lung cancer in Seoul National University Hospital pathology department (Park et al. "EGFR gene copy number in adenocarcinoma of the lung by FISH analysis: investigation of significantly related factors on CT, FDG-PET) , and histopathology. "Lung Cancer. 64 (2): 179-186 (2009)).

2.4. Drug Reactivity Criteria

The standard for determining drug reactivity is RECIST (The Response Evaluation Criteria in Solid Tumors), which measures drug reactivity using the major axis (The longest diameter measured in the axial-oblique plane of acquisition) of MRI images before and after drug administration. To predict. In the 21 patients used for FISH Validation in this example, the length of the major axis of the tumor was measured on the MRI image before and after the drug administration. And if it is more or more, it is not reactive. This can be used to quantify whether the selected two marker-based prediction models can accurately predict drug-reactive and non-reactive groups. FISH results are shown in the following Table 5, FIGS. 3A-3J and 4A-4J.

TABLE 5

sID RECIST 17q21.32 -33 Amplification 17p12 deletion Major axis pre-size Major axis post-size Major axis Size change S 060000903 SD 0 -One 7.5 6 -1.5 S 060004351 PR One 0 4.8 1.8 -3 S 060004464 SD 0 -One 5.1 5.5 0.4 S 060006236 PR 0 -One 4.9 1.8 -3.1 S 060006296 PR One 0 3.2 1.6 -1.6 S 060008606 SD One 0 5.7 5.7 0 S 060010590 PR One 0 4.8 2.3 -2.5 S 060014716 SD One -One 2.2 1.6 -0.6 S 060015049 PR 0 0 5.9 3.4 -2.5 S 060015994 PR 0 0 7.6 4.9 -2.7 S 060019506 PR 0 -One 3 2.1 -0.9 S 060023954 PR 0 0 3.2 1.6 -1.6 S 060028579 PR 0 0 3.8 2.2 -1.6 S 060031259 PR 0 0 5.5 3.4 -2.1 S 060042391 SD One 0 6.8 3.4 -3.4 S 060043817 PR 0 0 4.8 1.9 -2.9 S 070012306 PR 0 0 6 1.2 -4.8 S 070035644 PR 0 0 10.1 4.9 -5.2 S 070036677 PR 0 0 5.1 1.8 -3.3 S 070038079 PR 0 0 9 4 -5 S 080006841 SD 0 -One 5.2 5.2 0

sID: Sample ID

SD: Stable disease (determined not reactive)

PR: Partial Response

2.5. 17 using data mining algorithm p12  Loss with 17 q21 Drug Reactivity Predictive Testing of .32-33 gain

In the present embodiment, as an algorithm widely used for prediction modeling, Artificial Neural Net, Logistic Regression, and Decision Tree (above, WEKA data mining system http://www.cs.waikato.ac.nz/ml/weka/) are all used. Using the two markers, the model was constructed and predicted. Prediction accuracy of these two markers was 80.9524%, Sensitivity: 66.7%, and Specificity: 86.7%. As there are currently no criteria for predicting drug responsiveness, the predictive power of the two regions obtained from the validation data set is significant for both clinicians and patients.

1 schematically shows a cancer tissue sampling process.

FIG. 2 shows genomic regions that are significantly correlated with the reduced volume ratio detected to the extent of tumor volume reduction.

3A-3J show genome examples of breast cancer patients showing a loss of the 17p12 region.

4A-4J show genome examples of breast cancer patients showing gains in 17q21.32-33 sites.

Figure 5 shows the FISH results of breast cancer patients showing a defect in the 17p12 region.

Figure 6 shows the FISH results of breast cancer patients showing amplification of the 17q21.32-33 site.

Claims (4)

Genomic DNA fragments 17q21.32-33 shown in Table 1 below; 20 to 100 bp oligonucleotides located consecutively within the genomic DNA fragment; 100 bp to 100 kbp of cDNA derived from said genomic DNA fragment; And at least one probe selected from the group consisting of the genomic DNA fragment, oligonucleotide, and the complement of cDNA, Compositions for predicting drug reactivity to at least one selected from the group consisting of docetaxel and doxorubicin in breast cancer patients: TABLE 1 Position in the human genome Size (bp) chromosome Cytoband start End 17 17q21.32-33 41,900,001 47,600,000 5.7 Mbp The method of claim 1, The probe may be a genomic DNA fragment comprising BAC (Bacterial Artificial Chromosome), HAC (Human Artificial Chromosome), TAC (Transformation competent Artificial Chromosome), YAC (Yeast Artificial Chromosome), PAC (P1-derived Artificial Chromosome), P1 (phage), It is prepared by inserting into a vector selected from the group consisting of plasmid and cosmid (cosmid), the composition for predicting drug reactivity of breast cancer patients. Comparing the number of copies of the 17q21.32-33 region of human chromosome 17 in the DNA sample obtained from the breast cancer patient with the number of copies of the chromosomal portion of the normal DNA sample; When the number of copies of the 17p21.32-33 region of chromosome 17 in a DNA sample obtained from a breast cancer patient was increased than that of a normal DNA sample, the patient was determined to be non-responsive to at least one selected from the group consisting of docetaxel and doxorubicin. Characterized by Method for predicting drug reactivity in breast cancer patients. The method of claim 3, The copy number comparison is performed by one or more selected from the group consisting of PCR, array-based analysis, and fluorescence analysis, Method for predicting drug reactivity in breast cancer patients.

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