WO2005003352A1 - Procede d'evaluation de la sensibilite aux taxanes - Google Patents

Procede d'evaluation de la sensibilite aux taxanes Download PDF

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WO2005003352A1
WO2005003352A1 PCT/JP2004/009692 JP2004009692W WO2005003352A1 WO 2005003352 A1 WO2005003352 A1 WO 2005003352A1 JP 2004009692 W JP2004009692 W JP 2004009692W WO 2005003352 A1 WO2005003352 A1 WO 2005003352A1
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responsive
sample
genes
expression
gene
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PCT/JP2004/009692
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Japanese (ja)
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Kikuya Kato
Shinzaburo Noguchi
Kyoko Koizumi
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Taisho Pharmaceutical Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for determining the responsiveness of an individual to taxanes, and a probe set and a kit used for the method.
  • Docetaxel is one of the most effective anticancer drugs for the treatment of cancer, especially breast cancer
  • Taxanes such as docetaxel (taxane compounds) act by inhibiting microtubule dynamics, thereby arresting cells in the M phase of cell division and subsequently activating a program of apodosis.
  • docetaxel Taxanes
  • Taxanes act by inhibiting microtubule dynamics, thereby arresting cells in the M phase of cell division and subsequently activating a program of apodosis.
  • Jordan, MA et al. Current medicinal chemistry. Anti-cancer agents, 2002, 3 ⁇ 4 2, p. 1-17; Rao, S. et al., Journal of the National Cancer Institute, 1992, vol. 84, p. 785-788; Schiff, PB et al., Proceedings of the National Academy of Sciences of the United States of America, 1980, Vol. 77, p. 1561-1565; Stein, CA, Seminars in oncology, 1999, Vol. 26, p. 3-7).
  • taxanes are very effective anticancer drugs, it is known that about half of cancer patients do not respond to chemotherapy with taxanes and only produce side effects. Being able to predict a patient's responsiveness to a particular chemotherapy would allow for better treatment choices and rescue the patient from unnecessary side effects.
  • the present inventors studied the gene expression in the responsive group and the non-responsive group using the expression profile method in order to determine in advance the responsiveness of the patient to the taxanes.
  • a sample was obtained from human breast tumor tissue by biopsy before docetaxel treatment, and the response of the sample to treatment was clinically evaluated based on whether or not the tumor size was reduced.
  • gene expression profiling in biopsy samples was performed by high-throughput RT-PCR technology.
  • a sample derived from breast cancer that shows responsiveness to treatment (where treatment is effective) hereinafter referred to as a responsive sample
  • a sample that does not show responsiveness (where treatment is ineffective) hereinafter called non-responsive sample
  • the present invention provides the following (1) to (13).
  • Expression of 10 or more genes was detected in a sample derived from the individual, and Determining whether the sample is a responsive or non-responsive sample to taxanes.
  • T245 Homo sapiens T245 protein (T245) mRNA 0.324423328 0.03802 N
  • the breast cancer tissue is a primary breast cancer tissue or a locally recurrent breast cancer tissue.
  • a probe set comprising a probe for detecting the expression of 10 or more genes selected from the genes listed in Table 1 for determining the responsiveness to taxanes in a sample.
  • a kit for detecting taxane responsiveness comprising the probe set according to (5) to (7) or the microarray according to (8).
  • Taxane containing a drug listed in Table 1 that contains an agent that suppresses the activity of the expression product of one or more genes that are highly expressed in a non-responsive sample Pharmaceutical compositions for administration to non-responsive patients.
  • FIG. 1 shows the classification accuracy of the method of the invention as a function of the number of genes used.
  • ⁇ indicates the result of gene selection by recursive feature elimination and prediction based on support vector machines.
  • the dotted line shows the accuracy of the cross-validation performed after selecting genes from 44 samples.
  • the X axis is the number of reporters.
  • FIG. 3 shows the induction of apoptosis by docetaxel in MCF-7 cells transfected with the control and the Reddotas gene.
  • Control gene (chosen at random
  • MCF-7 cells transfected with a gene encoding a fusion protein of 5 amino acids and GFP ⁇
  • MCF-7 cells transfected with a gene encoding thioredoxin ⁇
  • daltathione-S- The MCF-7 cells transfected with the gene encoding transferase Pi1 (horizon) and the MCF-7 cells transfected with the gene encoding peroxyredoxin ( ⁇ ) were compared with the various cells shown in the figure.
  • the Y-axis shows the ratio of surviving cells to untreated controls. The experiment was performed twice. '' Best mode for carrying out the invention
  • the present invention relates to a method for determining the responsiveness of an individual to taxanes, comprising detecting the expression of 10 or more genes listed in Table 1 for a sample derived from the individual, and determining the expression of the taxane based on the detection result. And a method for determining whether the sample is a responsive sample or a non-responsive sample to the sample.
  • the term “individual” refers to a human cancer patient or a human individual at risk of cancer, and particularly a human primary or recurrent breast cancer patient or a human individual at risk of breast cancer. Say.
  • examples of the taxane include paclitaxel, docetaxel, and pharmaceutically acceptable and clinically effective derivatives thereof, and are not particularly limited.
  • Paclitaxel and docetaxel are available under the trade names Taxol and Taxotere, respectively, for example, from Bristol-Myers.
  • responsiveness refers to a response based on the following criteria by reducing the size of a tumor after chemotherapy with taxanes:
  • Progression of disease An increase of 25% or more in tumor size or appearance of new lesions.
  • a sample evaluated as CR or PR is defined as a “responsive” sample, and a sample evaluated as NC or PD is defined as a “non-responsive” sample.
  • the method of the present invention comprises the steps of: expressing the expression of a particular gene in a responsive sample (in a training set) and a non-responsive sample; Using information correlated with clinical observations of tumor size after chemotherapy with the chemotherapy, this sample is actually better than the chemotherapy with the taxanes, based on the expression of the same gene in the sample of interest (for example, in a certification set). It is for predicting whether or not a large reduction in tumor size can be obtained.
  • the sample may be a tissue obtained by incision biopsy or suction core-dollar biopsy from the individual, or may be a tissue that has been collected from the individual and cultured and preserved.
  • the tissue can be either a primary cancerous tissue or a recurrent cancerous tissue.
  • the sample is a human breast cancer tissue.
  • the gene group described in Table 1 was analyzed for gene expression in breast cancer samples responsive to taxanes and in non-responsive breast cancer samples. Are the most effective genes in predicting whether a breast cancer sample is responsive or non-responsive to taxanes before chemotherapy. The present inventors predict with high precision whether a sample is responsive or non-responsive to taxanes by detecting the expression of any 10 or more genes from this gene group. I found that I can do it.
  • the number of genes to be used may be 10 or more, preferably 20 or more, more preferably 40 or more, still more preferably 60 or more, and still more preferably 80 or more. As described above, most preferably, all 85 genes shown in Table 1 may be used.
  • the 10 or more genes to be used are not particularly limited, but the difference between the expression levels is large using the permutation p-value or the absolute value of the signal-to-noise ratio (SNR) as an index. It is particularly preferable to select them in order from the ones. In addition, this may detect the expression of other genes at the time of detection, and does not particularly mean that only the genes listed in Table 1 are detected.
  • the gene group described in Example 7 in Table 7 is a gene group showing significant expression selected by the permutation test, and includes all the genes described in Table 1. Genes not included in 1 may be used for detection.
  • the responsiveness to taxanes was improved by the expression of 10 or more, 20 or more, 40 or more, 60 or more, and 80 or more genes listed in Table 7 as in the method of the present invention. It is possible to decide. However, the genes in Table 1 are not Since it was narrowed down to high genes, it was selected from about 1100 genes that had good amplification in the PCR reaction, and the best results could be obtained.
  • the expression of the gene is not particularly limited, but the base of the gene to be detected.Hybridization reaction is performed using a probe having a sequence complementary to the sequence, and the presence or absence of hybridization is detected. And can be detected by detecting Z or level.
  • the nucleotide sequences of the genes described in Table 1 those registered in databases such as GenBank can acquire nucleotide sequence information based on the registration numbers described in Table 1.
  • those not registered in the database are listed as SEQ ID NOS: 1 to 7 in the sequence listing. Therefore, for each gene, a probe having a base sequence consisting of, for example, 15 to 100 bases, preferably 20 to 50 bases, which is complementary to the base sequence, is available in the art.
  • reaction may be performed separately for each gene.However, by using a microarray or the like in which individual probes are immobilized on a solid support, the expression of multiple genes can be detected simultaneously. it can.
  • the expression product Prior to detection, the expression product can be amplified using a PCR reaction or the like to increase the detection sensitivity.
  • RNA is purified from the sample by a method generally used in this field, and then a specific primer is designed for each gene, and a PCR reaction is performed.
  • the expression level of the gene can be measured by using these primers and real-time PCR.
  • the responsiveness is not particularly limited, but for the 10 or more detection target genes selected from the genes listed in Table 1, As described in the example of the above, by calculating the sum of weighted voting of genes using the weighted voting algorithm and calculating the prediction strength using the prediction strength 0 as a threshold, if the prediction strength is greater than 0, If it is less than 0, it can be determined as a non-responsive sample. Alternatively, it is preferable to prepare a standard expression pattern in a responsive sample and a non-responsive sample in advance, and determine the expression pattern based on the pattern.
  • the responsive sample shows a specific expression pattern, that is, the genes listed in Table 1 that are highly expressed in the responsive sample (Table 3 below) have relatively high expression levels
  • the expression level of a gene that is highly expressed in the sample (Table 4 below) is relatively low
  • the sample can be determined to be a responsive sample.
  • the non-responsive sample shows a characteristic expression pattern, that is, the gene expressed in the non-responsive sample in the genes shown in Table 1 has a relatively high expression level and the responsive sample has a high expression level.
  • the expression level of the expressed gene is relatively low, the sample can be determined to be a non-responsive sample.
  • the K-nearest neighbor method (k-nearest neighbor (k- ⁇ ), Pomeroy, SL et al., Prediction of central nervous system embryonal tumour outcome based on gene expression.Nature 415, 436-442 (2002) ) Is preferably used.
  • Probes derived from 0 or more, 40 or more, 60 or more, 80 or more, or 85 genes, or a support comprising these probes together with probes derived from other genes A microarray was prepared by immobilizing the sample on the
  • the expression pattern of each sample can be prepared by appropriately labeling the mRNA extracted from the Z or non-responsive sample, or the cDNA or PCR product obtained by reverse transcription thereof, and then hybridizing it. Then, using a microarray having probes in the same arrangement, the mRNA extracted from a sample (sample) for determining the response, or the cDNA or PCR product obtained by reverse transcription of the extracted mRNA is hybridized, and By examining the expression pattern, it is possible to determine whether the target sample is responsive or non-responsive.
  • the detection of a gene using a microarray is known in the art, and various commonly used detection means can be used. In carrying out the method of the present invention, detection by the above-described fluorescent dye is particularly limited. Not a thing.
  • the present invention also provides a probe set comprising a probe for detecting the expression of 10 or more genes selected from the genes listed in Table 1, for determining the responsiveness to taxanes in a sample.
  • the probe set includes probes respectively corresponding to any of 10 or more genes selected from the group of genes described in Table 1, but is preferably 20 or more, and more preferably 4 or more, described in Table 1. It contains a probe corresponding to 0 or more, more preferably 60 or more, more preferably 80 or more, and most preferably all of the 85 genes shown in Table 1.
  • the 10 or more types of genes to be selected are not particularly limited, but it is particularly preferable to select them in descending order of expression level using the p-value or the absolute value of SNR as an index.
  • One form of the probe is a primer for PCR amplification of the genes listed in Table 1.
  • probe specifically hybridizes with the genes listed in Table 1, and has, for example, a nucleotide sequence consisting of 15 to 100, preferably 20 to 50 bases. It is.
  • the term “specifically hybridizes” refers to, for example, being capable of hybridizing under high stringency conditions, and particularly being complementary. Those skilled in the art can appropriately set conditions for specific hybridization depending on the length of the probe, the GC content, and the like.
  • the present invention further provides a probe for the above probe set, Provide a microarray fixed at a given position.
  • the microarray include a DNA chip having probes immobilized on a substrate.
  • the present invention further provides a taxane responsive detection kit comprising the above probe set or the above microarray.
  • the above probe set, microarray and kit can be used to carry out the method of the present invention.
  • the present invention also relates to a gene described in Table 1, which suppresses the expression of one or more genes highly expressed in a non-responsive sample, that is, one or more genes described in Table 7.
  • a gene described in Table 1 which suppresses the expression of one or more genes highly expressed in a non-responsive sample, that is, one or more genes described in Table 7.
  • Provided is an antisense nucleic acid for administration to a patient who is not responsive to taxanes. ,
  • the antisense nucleic acid has a nucleotide sequence that is complementary to the nucleotide sequence of one or more of the genes listed in Table 1, which are highly expressed in a non-responsive sample, In addition, the expression of the nucleic acid can be suppressed.
  • the gene with accession number M24485 corresponding to number 10 in Table 1 is highly expressed ("N") in non-responsive samples.
  • An antisense nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of this gene (daltathione-S-transferase pi gene) and capable of suppressing the expression of the nucleic acid is introduced into, for example, a cancer cell.
  • the antisense nucleic acid preferably has, for example, a sequence complementary to, for example, a partial sequence of 14 or more consecutive bases of the base sequence of the gene with accession number M24485.
  • Antisense nucleic acids can be applied to cancer cells that are not responsive to taxanes, by DNA transfection methods such as the calcium phosphate method, lipofection method, electoral poration method, microinjection method, or viruses.
  • the gene can be introduced using a method known in the art, such as a gene introduction method including the use of a gene transfer vector.
  • a vector for expressing an antisense nucleic acid may be prepared using an appropriate retroviral vector, and then the expression vector may be introduced into a cell by contacting the cell with the cell in vivo or ex vivo.
  • the present invention also relates to the genes listed in Table 1, which are highly expressed in non-responsive samples.
  • Provided is a double-stranded RNA nucleic acid for suppressing the expression of one or more existing genes and for administering to a patient who is not responsive to taxanes.
  • double-stranded RNA that suppresses gene expression is particularly preferably RNA having an RNAi effect.
  • the RNAi RNA interference
  • a double-stranded RNA containing at least 10 consecutive nucleotides of RNA (base sequence) transcribed from a gene highly expressed in a non-responsive sample can be mentioned.
  • the details of RNAi are described, for example, in Experimental Medicine separate volume “RNAi Experiment Protocol” (Yodosha, Kazumasa Tahira et al.).
  • the double-stranded RNA nucleic acid can be administered to a cancer patient in the same manner as the antisense nucleic acid.
  • the present invention is further administered to a non-responsive patient to a taxane, which binds to an expression product of one or more of the genes listed in Table 1 and which is highly expressed in a non-responsive sample.
  • a taxane which binds to an expression product of one or more of the genes listed in Table 1 and which is highly expressed in a non-responsive sample.
  • the antibody may be any antibody that specifically binds to the above expression product and neutralizes its function or biological activity, and may be a polyclonal antibody or a monoclonal antibody. Furthermore, antibody fragments such as Fab and Fc may be used.
  • the antibody is preferably a human or humanized antibody.
  • the antibody of the present invention can be obtained, for example, using the above-mentioned expression product or a fragment thereof as an antigen.
  • the polyclonal antibody can be produced by a usual method of inoculating a host animal (eg, mouse, rat, egret, etc.) with an antigen and collecting serum.
  • Monoclonal antibodies can be produced by conventional techniques such as the hybridoma method.
  • the function or biological activity of the expression product can be suppressed.
  • the antibody of the present invention is combined with an inactive ingredient such as an immunogenic adjuvant or an additional active ingredient for therapeutic use, together with a stabilizer and an excipient, and after sterilization by filtration, a lyophilized product, Or it can be obtained as a stock in a stabilized aqueous preparation.
  • Administration can be performed by a method known in the art, such as subcutaneous injection, intraarterial injection, or intravenous injection. The dosage depends on the weight of the patient, Those skilled in the art can appropriately select them according to the tumor size, the administration method, and the like.
  • the present invention further relates to a non-taxane against the taxane, which comprises an agent that suppresses the activity of the expression product of one or more of the genes listed in Table 1 and that is highly expressed in a non-responsive sample.
  • a non-taxane against the taxane which comprises an agent that suppresses the activity of the expression product of one or more of the genes listed in Table 1 and that is highly expressed in a non-responsive sample.
  • Examples of the drug that suppresses the activity of the gene expression product include, but are not limited to, an antibody that binds to the expression product and suppresses its physiological activity, an enzyme inhibitor, and the like.
  • the present inventors have found several genes that regulate the intracellular redox environment in the gene groups shown in Tables 1 and 7. Then, it was confirmed that taxane-responsive cells transfected with these genes acquired non-responsive properties and became resistant to the action of taxanes. Therefore, the use of inhibitors against the activity of the proteins or peptides encoded by these genes is extremely effective in treating tumors that are not responsive to taxanes.
  • GSTP1 daltathione-S-transferase pi 1
  • glutathione peroxidase 1 glutathione peroxidase 1
  • thioredoxin glutathione peroxidase 1
  • peroxyredoxin 1 glutathione peroxidase 2
  • glutathione peroxidase 4 glutathione peroxidase 4
  • inhibitors for enzymes such as glutathione peroxidase-related protein 1 can be used for treating tumors such as breast cancer.
  • the responsiveness to taxanes is adjusted, responsiveness is imparted to non-responsive tumors, and taxanes that are originally non-responsive to taxanes are administered.
  • the enzyme inhibitor may be administered alone to a taxane non-responsive individual, or may be administered in combination with a taxane.
  • the composition can be administered as a pharmaceutical composition in combination with excipients, carriers, buffers and the like usually used in the pharmaceutical composition.
  • one or more enzyme inhibitors can be combined and provided as a therapeutic kit for a taxane non-responsive tumor.
  • Table 2 shows the age, menopausal status, and cancer status of 44 patients as a learning set and 26 patients as a validation set from which the samples were collected.
  • Docetaxel (6 Omg / m 2 , intravenous injection every 3 weeks) as chemotherapy for each of the above patients, 4 cycles before surgery for patients with primary breast cancer, locally recurrent In the case of breast cancer patients, administration was continued until disease progression began. Patients with locally recurrent breast cancer receive docetaxel as the first chemotherapy after recurrence and no adjuvant taxane treatment.
  • Progression of disease (PD) 25% increase in tumor size or appearance of new lesions.
  • a sample classified as CR or PR is defined as a responsive sample
  • a sample classified as NC or PD is defined as a non-responsive sample.
  • Forty-two patients obtained 22 responsive samples and 22 non-responsive samples to treatment.
  • RNAs derived from 12 breast cancer tissues different from those used in Example 1 (Iwao, K. et al., Hum. Mol. Genet. 11, 199-206 (2002) )) was used to prepare a 3′-terminal cDNA library.
  • ATAC-PCR PCR primers for the reaction were set up. Two of these adapters were used as a control with a mix of 78 primary breast cancer mRNAs. ATAC-PCR is an improved method of quantitative RT-PCR discovered by the group of the present inventors.
  • RT-PCR is an advantageous method for analysis on clinical samples because it has a wider dynamic range of detection and requires less RNA than DNA microarrays.
  • the expression level of the 2453 gene in breast cancer tissues was measured by ATAC-PCR. Amplification products were separated by ABI PRISM 3700 DNA analyzer (Applied Biosystems) or ABI PRISM 3100 Genetic analyzer (Applied Biosystems). Next, for each gene, the relative expression level compared to the control was calculated. The obtained data matrix was normalized with a median by a method similar to the method used in DNA microarrays (vant Veer. LJ. Et al., Nature 415, 530-536 (2002)) and converted to a logarithmic scale.
  • the expression data of the 2453 gene in 44 samples was obtained as a relative ratio to the control by ATAC-PCR.
  • a control a mixture of RA purified from 78 breast cancer tissues was used. After removing 235 genes whose number of missing values was more than 20% of all samples, only 2218 genes whose number of missing values was less than 20% were used for gene selection. As a result, it is possible to focus on the gene with the largest amount of information.
  • 1125 genes with good PCR reactions were selected from 2218 genes and used, but there was no bias in the function or properties of the genes.
  • the gene expression crude data was converted so that the median value of each sample was 1. Values less than 0.05 were converted to the minimum limit value of 0.05.
  • the weighted voting algorithm is a method of predicting the sum of weighted votes of each gene.
  • the weight used is the signal-to-noise ratio (SNR) calculated from the data of the samples (44 cases in this example) included in the training set. If the average expression level of the responsive sample in the learning sample of gene g is ⁇ R g and the standard deviation is ⁇ R g .
  • the average expression level of the non-responsive sample is N g and the standard deviation is a N g , the SNR Can be obtained by the following equation (1).
  • the weighted voting of gene g can be obtained by the following equation 2.
  • Weighted vote of gene g ( ⁇ ⁇ ) X, (Equation 2)
  • the weighted voting of the gene g in the sample can be calculated. This weighted vote is calculated for all genes.
  • Prediction strength a measure of the degree of “prediction”, is defined by Equation 4 below. If the value is large, the response sample is predicted, and if the value is small, the sample is non-responsive. For example, the threshold value of the prediction strength is set to 0. If the threshold value is larger than ⁇ , the response sample can be predicted.
  • VR + Leave-one-out cross-validation procedure without information leakage is as follows. First, one sample was extracted and the remaining 43 samples were analyzed for the individual 1125 genes using SNR, PPT (50,000 random exchanges), or RFE to determine the logarithm of docetaxel sensitivity and expression ratio. The correlation between them was calculated. Next, the genes were sorted by the absolute value of each value. Then, based on the set of top ranked genes, we predicted the outcome of one sample that was omitted using the WV, k-NN, or SVM algorithm. WV: The threshold value of predicted strength for classifying responsive and non-responsive samples was 0. The k value of k-NN (nearest neighbor) is 1.
  • the strict leave-one-out cross-paridation which includes both the gene selection step and the prediction algorithm, shows that the WV algorithm generally gives good results, with the highest accuracy when using 85 genes (72. 7%) ( Figure 1). However, even when 10 or more genes are used, an accuracy of about 65% can be obtained, which is effective for simple clinical judgment. Genes were selected in descending order of expression level using the absolute value of SNR as an index. In cancer classification, cross-validation is often performed only with a prediction algorithm after selecting genes from all data sets. This type of cross-validation overestimates the accuracy of the prediction, but is useful for setting an upper bound. In this example, WV evaluation accuracy generally exceeded 90%, and good results were obtained with 10 or more genes (FIG. 1). From these analysis results, 85 genes listed in Table 1 were selected as a gene set for diagnosis by weighted-voting, and the accuracy was predicted to be 70 to 90%.
  • the numbers of the genes are the same as those in Table 7 below.
  • the registration number indicates the registration number in the GenBank database. Genes with registration numbers can access the database via the Internet, for example, to obtain nucleotide sequence information. Genes for which registration numbers are not described (gene numbers 4, 57, 59, 112, 121, 122, and 125) are gene sequences newly discovered by the present inventors. And its base sequence is shown in the sequence listing as SEQ ID NOS: 1 to 7, respectively. 'In addition, the p value in the SNR and permutation test was shown for each gene. “RZN” indicates whether each gene is a gene that is highly expressed in a responsive sample (R) ⁇ a gene that is highly expressed in a non-responsive sample (N). For convenience, the genes listed in Table 1 are divided into genes (R) that are highly expressed in responsive samples and genes (N) that are highly expressed in non-responsive samples, and are listed in Tables 3 and 4, respectively.
  • RNB6 Homo sapiens RNB6 (RNB6) mRNA, complete cds.0.36547013 0.02874 R serine (or cysteine) proteinase inhibitor, clade A (alpha-1
  • OAV solute carrier family 25 mitochondrial carrier; phosphate carrier
  • T245 Homo sapiens T245 protein (T245) mRNA 0.324423328 0.03802 N
  • the expression ratio of the determined 85 marker genes (Table 1) in 44 samples was used to calculate the predicted strength of 26 samples for verification. Responsiveness sample (predicted intensity The threshold of the prediction strength, which is classified into “strong”) and non-responsive samples (predicted strength “weak”), was set to 0.
  • the expression level of the 85 genes was measured by ATAC-PCR in each of the samples obtained from the patients, and the response to docetaxel was predicted.
  • Table 6 shows the relationship between the predicted strength and the actual response to docetaxel treatment in the validation set. The numbers in the table indicate the number of patients.
  • the numbers of the genes are the same as those in Table 1.
  • Table 7 is sorted in ascending order of p-value.
  • primers that can be used for PCR amplification of each gene are shown in SEQ ID NOS: 8 to 155.
  • non-responsive samples show increased expression of genes that regulate the intracellular redox environment.
  • genes include daltathione-S-transferase pi 1 (GSTP1) (gene number 10), daltathione peroxidase 1 (gene number 86), thioredoxin (gene number 87), peroxyredoxin 1 (gene number 87) Genes encoding thioredoxin peroxidase 2) (gene number 85), glutathione peroxidase 4 (gene number 110), and daltathione peroxidase-related protein 1 (gene number 16).
  • redox genes genes encoding these redox-related peptides or proteins (hereinafter referred to as “redox genes”) are abnormally highly expressed in samples that do not respond to docetaxel. It was strongly suggested that it was one of the redox genes
  • the expression of the redox gene was related to docetaxel resistance by transfection experiments.
  • Glutathione - S- transferase pi (GenBank Accession No. M24485, gene number 1 0), Chioredokishin (GenBank Accession No. BC00337 7, gene number 8 7), ⁇ Pi
  • the three genes encoding peroxyredoxin (GenBank accession number X67951, gene number 85) were cloned as fusion proteins with GFP (green fluorescent protein) under the control of the cytomegalovirus promoter.
  • GFP green fluorescent protein
  • MCF-7 breast cancer cells were cultured in thigh M (SIGMA, St Louis, M0) supplemented with 10% FBS (Dainippon Pharm.) And ant imycot ic-ant imytotic (GIBCO BRL). ° 5% Rei_0 grown in 2 humidified incubator in C, and divided two times a week.
  • Transfected cells were grown on 24 multi-well culture dishes and treated with various concentrations of docetaxel. Twenty-four hours later, cells are fixed with 4% paraformaldehyde in PBS, stained with -TMR-: red (Roche Molecular Biochemicals, Mannheim, Germany), and cell death is detected with TUNEL Acetsee, which detects fragmentation of chromosomal DNA. Judged.
  • the method of the present invention can grasp most of the inherent properties of cancer tissues with respect to responsiveness to taxanes, and provides clinically useful guidelines for deciding treatment methods for human cancer. Can be provided. Further, the results of the present inventors show that a novel anticancer agent can be designed to provide a responsive property to a non-responsive sample by inhibiting daltathione and Z- or thioredoxin-based enzymes, and to assist the effects of taxanes. It is suggested.
  • Breast cancer genes in well-designed clinical trials Expression profiling is very important for clinical and pharmaceutical development. Responsiveness to a drug varies from individual to individual, and predicting this will allow for treatment decisions, drug development, and medications only for sensitive patients.
  • SNPs single nucleotide polymorphisms
  • Glutathione and thioredoxin systems have previously been studied separately, but the results of the present inventors suggest that both of these systems cooperate in resistance to taxanes. Is done. Although the details of the mechanism of these enzymes are unknown, protection from oxidative stress induced by taxanes, due to the presence of peroxidase, is the most likely mechanism.
  • the responsiveness to an anticancer drug depends not only on the properties inherent in the cancer tissue but also on various host-side conditions such as drug metabolism. For this reason, very accurate predictions are generally difficult. Therefore, the method of the present invention grasps most of the inherent properties of cancer tissues with respect to responsiveness to taxanes, and can serve as a clinically useful guide when deciding a treatment method for cancer. it is conceivable that. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

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Abstract

La présente invention concerne l'évaluation préalable de la sensibilité d'un patient aux taxanes. A cet effet, on commence par détecter les expressions d'au moins 10 gènes énumérés au Tableau 1 dans un échantillon prélevé chez le sujet considéré. On détermine alors, sur la base des résultats de détection, si l'échantillon est sensible ou non aux taxanes.
PCT/JP2004/009692 2003-07-01 2004-07-01 Procede d'evaluation de la sensibilite aux taxanes WO2005003352A1 (fr)

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DE102004058201A1 (de) * 2004-12-02 2006-06-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zur Übertragung von Bauelementen auf eine Oberfläche
EP2047864A1 (fr) * 2006-06-16 2009-04-15 Taisho Pharmaceutical Co., Ltd Utilisation d'un inhibiteur de l'expression du gène rpn2
JP2012173278A (ja) * 2011-02-24 2012-09-10 Sysmex Corp 癌細胞のタキサン系抗癌剤感受性判定方法、それを実現するためのコンピュータプログラム及び装置

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CN102548549A (zh) * 2009-08-25 2012-07-04 细胞研究有限公司 利用外代谢转换剂(辅酶q10)治疗肉瘤的方法
JP6425483B2 (ja) * 2013-09-30 2018-11-21 国立大学法人東京工業大学 細胞内の酸化還元状態をモニターするための蛍光タンパク質、ｄｎａ、ベクター、形質転換体、及び方法
CN115803035A (zh) * 2020-06-15 2023-03-14 株式会社Kortuc 抗癌疗法的增敏剂

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TIMMS, K.M. ET AL.: "130kb of DNA sequence reveals two new genes and a regional duplication distal to the human iduronate-2-sulfate sulfatase locus", GENOME RES., vol. 5, 1995, pages 71 - 78 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004058201A1 (de) * 2004-12-02 2006-06-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zur Übertragung von Bauelementen auf eine Oberfläche
EP2047864A1 (fr) * 2006-06-16 2009-04-15 Taisho Pharmaceutical Co., Ltd Utilisation d'un inhibiteur de l'expression du gène rpn2
EP2047864A4 (fr) * 2006-06-16 2010-12-01 Taisho Pharmaceutical Co Ltd Utilisation d'un inhibiteur de l'expression du gène rpn2
US8106024B2 (en) 2006-06-16 2012-01-31 Taisho Pharmaceutical Co., Ltd. Method of treating cancer with an RPN2 gene expression inhibitor
JP5131927B2 (ja) * 2006-06-16 2013-01-30 大正製薬株式会社 Ｒｐｎ２遺伝子発現抑制剤の用途
JP2012173278A (ja) * 2011-02-24 2012-09-10 Sysmex Corp 癌細胞のタキサン系抗癌剤感受性判定方法、それを実現するためのコンピュータプログラム及び装置

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