KR101748867B1 - Automated system for prognosing or predicting early stage breast cancer - Google Patents
Automated system for prognosing or predicting early stage breast cancer Download PDFInfo
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Abstract
The present invention relates to an automated system for predicting the prognosis of early breast cancer, and more particularly, to an automated system for predicting the prognosis of early breast cancer. More particularly, the present invention relates to an automated system for predicting breast cancer prognosis, , Apparatus and computer readable media suitable for the method. Since the system of the present invention can predict and diagnose the prognosis of breast cancer by an automated process, it can be usefully used for judging the necessity of chemotherapy and for suggesting a clue to the direction of breast cancer treatment in the future.
Description
The present invention relates to an automated system for predicting early breast cancer prognosis, and more particularly, to a method for predicting the prognosis of breast cancer patients, To an apparatus and a computer-readable medium suitable for the method.
Breast cancer can be diagnosed automatically, and the importance of self-diagnosis is often promoted, and it is often found early on. It was difficult to determine the postoperative chemotherapy for these early breast cancer patients. Although pathologic observation can predict the prognosis roughly, it is difficult to standardize and quantify the results of observation, and the reliability of prediction of prognosis is low. In actual clinical trials, most early breast cancer patients are recommended for chemotherapy. Because of the nature of chemotherapy, the suffering suffered by the patient is very large and economic expenditure is required. In early breast cancer, it is estimated that more than half of the patients do not need chemotherapy. Therefore, by analyzing the characteristics of early breast cancer and predicting the prognosis of the patient and reducing unnecessary chemotherapy, the quality of life of the patient will be greatly helped. As the information on the expression level of tens of thousands of genes of breast cancer can be obtained at a time using microarray, studies are being actively carried out to classify breast cancer at the molecular level and to clarify the mechanism of development and development of cancer have. Predicting the prognosis of patients with early breast cancer is important in clinical practice, and finding genes that predict prognosis using microarrays has already begun in the early 2000s. Despite the high cost of microarray studies, a significant number of expression profiles for breast cancer tissues have been produced and disclosed to researchers. In 2002, 70 survivors of breast cancer tissues and 10 years of follow-up were analyzed to determine the prognosis of 70 breast cancer tissues, and then dozens of prognostic genes were published, some of them PLoS Biol 2 (2): p. E7 (2004); van, et al., 2004), which has already been commercialized and used in clinical practice (Chang, HY, et al., Gene expression signature of fibroblast serum response predictions NJ et al., Mediastin, J., et al., " De Vijver, MJ, et al., A gene-expression signature as a predictor of survival in breast cancer. Gene expression profiles to predict distant metastasis of lymph node-negative primary breast cancer. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415 (6871): 530-536 (2002); Wang, Y., et al. Lancet 365 (9460): 671-679 (2005); Buyse, M., et al., Validation and clinical utility of a 70-gene prognostic Paik, S., Development and clinical utility of a 21-gene recurrence score prognostic assay in patients with early breast cancer. J Natl Cancer Inst., 98 (17): 1183-92 cancer treated with tamoxifen. Oncologist 12 (6): 631-635 (2007); Paik, S., et al., A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351 (27): 2817-2826 (2004); Sotiriou, C., et al., Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 98 (4): 262-72 (2006); Pawitan, Y., et al., Gene expression profiling sparing early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts. Breast Cancer Res 7 (6): R953-964 (2005); Miller, LD, et al., An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc Natl Acad Sci USA, 102 (38): 13550-13555 (2005); Bild, AH, et al., Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 439 (7074): 353-357 (2006); Teschendorff, AE, et al., A consensus prognostic gene expression classifier for ER positive breast cancer. Genome Biol 7 (10): R101 (2006); Desmedt, C., et al., Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series. Clin Cancer Res 13 (11): 3207-3214 (2007)). Mammaprint (Agendia) and Oncotype DX (genomic health) are currently used in clinical practice, but they are still used as a reference for prognosis (van de Vijver, MJ, et al., A (2002); Paik, S., et al., A multigene assay to predict recurrence of tamoxifen-treated, node N Engl J Med 351 (27): 2817-2826 (2004)).
In addition, these methods often fail to provide reliable results without being highly skilled. Oncotype DX can provide relatively meaningful results if the patient's sample is provided to the genomic health service according to the predetermined procedure in the central lab method. Thus, in the conventional prognostic prediction diagnosis method, there is a fear that an error may be generated in the prediction result of the prognosis according to the proficiency level of the experimenter. Therefore, in order to obtain a stable result, a method or an automated procedure in which the involvement of the experimenter is excluded is needed.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
Therefore, the present inventors have made intensive researches to develop a gene diagnosis system capable of diagnosing the prognosis and cancer treatment of early breast cancer patients using FFPE samples of tissues containing cancer cells of the patient. As a result, Data and clinical information were collected and analyzed to identify genes associated with prognosis prediction, and the present invention was completed by developing a suitable method, particularly an automated method.
It is therefore an object of the present invention to provide a novel method for predicting and prognosing a prognosis of a breast cancer patient.
Another object of the present invention is to provide an apparatus for calculating a prognostic value of a breast cancer patient.
It is yet another object of the present invention to provide a computer readable medium for calculating a prognosis prediction of a breast cancer patient.
In order to achieve the above object, the present invention provides a new method for predicting and diagnosing a prognosis of a breast cancer patient.
According to another aspect of the present invention, there is provided an apparatus for calculating a prognostic value of a breast cancer patient.
In order to achieve still another object of the present invention, the present invention provides a computer-readable medium for calculating a prognosis prediction value of a breast cancer patient.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide one of the skills with a general definition of the terms used in the specification of the present invention: Singleton et al ., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2ded.1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); And Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.
Hereinafter, the present invention will be described in more detail.
The present invention provides a method for calculating predicted breast cancer prognosis, comprising the following steps, from a sample of a patient, in order to provide information necessary for predictive prognosis of a breast cancer patient:
(a) treating a formalin-fixed paraffin-embedded (FFPE) sample containing a patient's breast cancer tissue with a buffer for paraffin removal and cell lysis;
(b) treating the sample of step (a) with a protease;
(c) removing protein or cell debris from the sample of step (b);
(d) obtaining RNA by treating the sample of step (c) with a DNA degrading enzyme;
(e) mixing the RNA with a primer set capable of amplifying the detection gene and an RT-qPCR premix;
(f) generating cDNA (complementary DNA) in the mixture of step (e);
(g) performing a PCR reaction using the cDNA as a template and using the primer set as a PCR primer;
(h) measuring the Cp value for the PCR reaction;
(i) standardizing expression level by standardizing Cp value for proliferation gene for breast cancer prognosis prediction diagnosis and Cp value for immunological gene for prediction of breast cancer prognosis to Cp value of standard gene;
(j) Standardized breast cancer prognosis prediction The lower the standardized expression level of the proliferation gene for diagnosis and the higher the normalized expression level of the immune gene for standardized breast cancer prognostic prediction diagnosis, the better the prognosis of breast cancer.
The method of the present invention is preferably an automated or semi-automated method. In the above, automation can be achieved by injecting a sample (sample); Relocation or movement of a substrate (e.g., tube, plate) that has been extracted, separated, or reacted; Reagent, buffer stock, replenishment; Means that all or most of the process, except equipment maintenance, takes place through means other than human (for example, a robot).
Each step will be described in detail below.
(a) is a step of treating a formalin-fixed paraffin-embedded (FFPE) sample containing breast cancer tissue of a patient with a buffer for paraffin removal and cell lysis.
The tissue obtained from the patient after biopsy is usually fixed with formalin (formaldehyde) or the like. The immobilized biological sample is generally dehydrated and embedded in a solid support such as paraffin, and the sample thus prepared is called an FFPE sample. Since the RNA on the FFPE sample is present in fixed cells and is fragmented or cross-linked by formalin, it is necessary to remove the paraffin and dissolve the fixed cells to elute the nucleic acid including the RNA in the cells.
In the present invention, the term " paraffin " comprehensively refers to a foraging medium of a biological sample used in all analyzes including morphological, immunohistochemical and enzymatic histochemical analysis. That is, the paraffin in the present invention may be a petroleum-based paraffin wax alone, and may be any one selected from the group consisting of all kinds of petroleum-based paraffin waxes, May be included. Herein, the petroleum paraffin wax refers to a mixture of hydrocarbons which are solid at room temperature derived from petroleum.
In the present invention, a sample of a breast cancer patient who has undergone FFPE treatment is preferably cut into a thickness of 5 to 10 mu m with a rotary microtome, and then a buffer (FFPE buffer, VERSANT tissue preparation reagents, Box 1, siemens) And incubated at 80 DEG C for 30 minutes.
The FFPE sample can be cut using a microtome for control of the amount of FFPE sample and ease of contact with the reagent. The cutting thickness is not limited to this, but is preferably 5 to 15 占 퐉. The cleaved FFPE sample is placed in an RNA extraction tube and the extraction process is carried out.
(b) is a step of treating a proteinase in the sample of step (a).
The proteins in the sample are decomposed and fragmented by the action of proteolytic enzymes. Preferably, the protease is protease K in the present invention. Protease K (proteinase K, EC 3.4.21.64) is a protease and serine protease found in the fungus Engyodontium album. The treatment with the protease K is preferably carried out under the conditions of 20 to 40 minutes, 45 to 70 ° C, more preferably 25 to 35 minutes, 60 to 65 ° C, most preferably 30 minutes, and 65 ° C. Treatment below the lower limit of the treatment conditions results in a lower efficiency of proteolytic degradation and ultimately reduces the efficiency of RNA isolation. Treatment above the upper limit reduces the RNA isolation efficiency due to degradation of the RNA during the separation process and increases the overall separation time, Fall off.
(c) is a step of removing protein or cell debris from the sample of step (b).
Proteins fragmented by the protease treatment in step (b) and cellular debris not degraded are separated and removed from nucleic acids such as RNA and DNA. The removal process can be carried out by binding magnetic beads and then imparting a magnetic force, obtaining a supernatant which is not precipitated or collected on one side, removing the precipitate through centrifugation, and obtaining only the supernatant.
(d) is a step of obtaining RNA by treating the sample of step (c) with a DNA degrading enzyme.
separation of the DNA and the nucleic acid containing the RNA is carried out through the separation up to the step (c). Treatment with DNA-degrading enzymes to isolate RNA only. The DNA-degrading enzyme may desirably be DNase I.
(e) is a step of mixing the RNA with a primer set capable of amplifying the detection gene and an RT-qPCR premix.
The isolated RNA is mixed with the primer set and RT-qPCR premix for the RT-qPCR reaction. A primer set can amplify a target gene, a reference gene, or a reference gene of interest. The RT-qPCR premix can be used for a reverse transcription reaction, a DNA polymerase (for example, a Tag polymerase) for PCR reaction, a dye (for example, a fluorescent dye) for quantitative detection of a PCR reaction, , DNTPs, and the like.
As used herein, the term " primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. Preferably, the primer is a deoxyribonucleotide and is a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.
The primer of the present invention may be an extension primer that is annealed to a target nucleic acid and forms a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which extends to a position where the immobilization probe is annealed, It occupies the area that is annealed.
The extension primer used in the present invention comprises a hybridization nucleotide sequence complementary to the first position of the target nucleic acid. The term " complementary " means that under certain annealing or hybridization conditions the primer or probe is sufficiently complementary to hybridize selectively to the target nucleic acid sequence and is substantially complementary and perfectly complementary , And preferably means completely complementary. As used herein, the term " substantially complementary sequence " as used in connection with a primer sequence is intended to encompass a complete sequence as well as a sequence that is comparable to that of the sequence to be compared, Inconsistent sequences are also included.
The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The suitable length of the primer is determined by a number of factors, such as the temperature, the application, and the source of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The term " annealing " or " priming " means that the oligodeoxynucleotide or nucleic acid is apposited to the template nucleic acid, which polymerizes the nucleotide to form a complementary nucleic acid molecule to the template nucleic acid or a portion thereof .
The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the above-mentioned nucleotide sequence, which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the above-mentioned nucleotide sequence, for example, by using a program for primer design (for example, PRIMER 3 program).
(f) is a step of generating cDNA (complementary DNA) in the mixture of step (e).
CDNA is synthesized from RNA by reverse transcription reaction. The enzyme, dNTP, and buffer necessary for synthesis are already contained in the RT-qPCR premix. The synthesis of the cDNA can be carried out before the PCR, preferably at 45 to 55 ° C for 15 to 30 minutes, more preferably at 50 ° C for 20 minutes, And the treatment exceeding the upper limit value leads to an increase in the overall separation time, resulting in a decrease in productivity.
Preferably, the PCR reaction proceeds immediately after the cDNA synthesis without any further treatment.
In the step (g), PCR is performed using the cDNA as a template and the primer set as a PCR primer.
PCR is carried out using the synthesized cDNA as a template. The PCR reaction can be performed by a method known in the art by quantitative PCR, but it can be preferably performed by fluorescence signal detection, and the fluorescence of a UPL probe (5'-FAM, 3'-Dark quencher) The amount of amplification of the PCR product according to the cycle can be detected in real time through the fluorescent material of the reporter dye generated when the quencher is removed.
(h) is a step of measuring the Cp value for the PCR reaction.
In quantitative PCR using fluorescent signals, the Cp value (equal to the Cp value, the Ct value, the cross point value) is much higher than the calculated baseline (the signal of the initial cycle of the PCR reaction) And a threshold value which is a level of the contact point. The Cp value can be used to calculate the original DNA copy number.
(i) is a step of standardizing the expression level by standardizing the Cp value for the proliferation gene for breast cancer prognosis prediction diagnosis and the Cp value for the immune gene for prediction of breast cancer prognosis to the Cp value of the standard gene.
In the present invention, the level of expression of an object to be detected may vary depending on the patient or sample, and thus standardization is necessary. The standardization is carried out through a difference from the expression level or expression level of a gene which can show a difference in basic expression level or expression level, preferably CTBP1 (C-terminal-binding protein 1), TBP (TATA-binding protein) The expression level of one to five genes (or the average of these expression amounts when a plurality of genes are selected) in HMBS (hydroxymethylbilane synthase), CUL1 (cullin 1) and UBQLN1 (Ubiquilin-1) For example. Synonyms and sequences for each gene can be found in Genbank.
Normalization can be calculated according to the following equation, and when a plurality of standard genes are present, standardized expression levels are calculated based on their average values, preferably geometric mean values.
Standardized expression level = 2 - ( target gene Cp value) / 2 - (standard gene average Cp value)
(j) shows that the lower the standardized expression level of the proliferation gene for standardized breast cancer prognosis prediction diagnosis and the higher the standardized expression level of the immune gene for the prediction breast cancer prognosis prediction diagnosis, the better the breast cancer prognosis is, .
The output algorithm can be derived according to the fitness of the results applied to the weave, logarithmic logistic and algebraic normal distribution. In the present invention, the final model according to the lognormal distribution is selected using Akaike's information criterion. The final estimated model for a frozen sample corresponding to the present invention instead of the FFPE sample is as follows.
log (T) = -0.689 x p.mean + 0.274 x i.mean + 3.219
According to the estimated model, the proliferation has a negative correlation (-0.689, p value = 2.47 x e-17) with the survival time (T) do. Conversely, i.mean has a positive correlation with survival time (0.274, p value = 3.69 x e-11), which means that the longer the immune response is, the longer the survival time is.
These results suggest that proliferation plays a decisive role in the prognosis of breast cancer, and the more active it is, the worse the prognosis, but the immune response acts as a defense mechanism against the rapid proliferation, which is consistent with conventional biological knowledge.
In the present invention, the proliferation gene for breast cancer prognosis prediction diagnosis is preferably a gene having three or more genes selected from the genes listed in Table 1, preferably 3, 4, 5, 6, 7, 8, 9, 11, and 12 genes, and p.mean applied to the algorithm is an arithmetic average value of the expression level of each gene. At this time, genes for which expression has not been detected can be excluded from the calculation.
In addition, the immune gene for prediction of breast cancer prognosis is preferably at least 3 genes selected from the genes listed in Table 2, preferably 3, 4, 5, 6, 7, 8, 9, And 12 genes, and i.mean applied to the algorithm is an arithmetic average value of the expression level of each gene. At this time, genes for which expression has not been detected can be excluded from the calculation.
In the present invention, the breast cancer may be invasive breast cancer, or stage I, II or III breast cancer. In addition, the breast cancer of the present invention may be an estrogen receptor positive (ER +).
In the present invention, " prognosis " refers to a prospect of future symptoms or progress judged by diagnosing a disease. Prognosis in cancer patients usually refers to the time of metastasis or survival within a period of time after cancer or surgery. Predictive prognosis (prognosis diagnosis or predictive diagnosis) is a very important clinical task because it provides clues to the future direction of breast cancer treatment, especially the chemotherapy of early breast cancer patients. Predictive prognosis also includes the patient's response to the disease treatment and the prediction of the course of treatment.
In the present invention, the sample may be a breast cancer tissue of a breast cancer patient. The breast cancer tissue may also contain some normal cells, and may be a formalin-fixed paraffin-embedded (FFPE) sample of breast cancer tissue, preferably comprising cancer cells of a patient.
Detection of the breast cancer prognostic predictive diagnostic marker of the present invention can be performed by PCR (polymerase chain reaction) amplification of the gene of interest. The detection of the gene of interest of the present invention is preferably a detection of the expression level of the gene of interest, more preferably the quantitative detection of the expression level of the gene of interest. In order to detect the expression level, mRNA isolation in the sample tissue and cDNA synthesis process in the mRNA may be necessary. In order to isolate mRNA, a method of isolating RNA in a sample known in the art can be used. Preferably, the sample is an FFPE sample, and thus may be a method for separating mRNA suitable for FFPE sample. As the cDNA synthesis process, a cDNA synthesis method known in the art using mRNA as a template can be used. Preferably, the detection of the breast cancer prognostic predictive diagnostic marker of the present invention is a quantitative detection of mRNA expression in the FFPE sample. Therefore, the detection may be performed by the mRNA separation method for the FFPE sample and the RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) method.
In the present invention, the detection may be a measurement of an mRNA expression level. Measurement of the expression level can be performed according to methods known in the art, but can be measured by an optical quantitative analysis system using a probe labeled with a reporter fluorescent dye and / or a quencher fluorescent dye. The measurement may be performed by commercially available equipment, for example, a system such as the ABI PRISM 7700 ™ Sequence Detection System ™, Roche Molecular Biochemicals Lightcycler, and accompanying software. Such measurement data may be expressed as a measurement value or a threshold value cycle (Ct or Cp). The point at which the measured fluorescence value is recorded as the first statistically significant point is the threshold cycle, which is inversely proportional to the initial value present as the template for the PCR reaction, so quantitatively Indicating that many detection targets exist.
On the other hand,
(a) means for separating the nucleic acid from the FFPE sample;
(b) a dispensing means for decomposing the nucleic acid, the buffer, and the reagent into a predetermined amount at a predetermined time at a predetermined position;
(c) Quantitative PCR (qPCR) means capable of performing PCR reaction and capable of measuring Cp value by detecting fluorescence signal;
(d) an arithmetic processing unit; And
(e) a computer readable medium executable by an arithmetic processing unit to convert the Cp value to an expression level, to calculate an average value of the expression level, and to calculate a predicted breast cancer prognosis according to a predetermined formula;
A predicted breast cancer prognosis prediction value calculation device.
The device of the present invention is an automated or semi-automated or automated / semi-automated device.
On the other hand,
the Cp value measured by the qPCR means is associated with the genetic information,
The ratio of the Cp value of the proliferative gene to the geometric mean of the Cp value of the standard gene for breast cancer prognosis prediction diagnosis was calculated to calculate the expression level of the proliferation gene for standardized breast cancer prognosis prediction diagnosis,
The ratio of the Cp value of the immune gene for the prediction of breast cancer prognosis to the geometric mean of the Cp value of the standard gene was calculated to calculate the expression level of the immune gene for standardized breast cancer prognosis prediction diagnosis,
To estimate the mean expression level of the proliferative gene for breast cancer prognosis prediction diagnosis and the average expression level of immune gene for breast cancer prognosis prediction diagnosis,
Provided is a computer readable medium for calculating a breast cancer prognosis prediction value in a positive correlation with an average expression level of an immune gene for prediction of breast cancer prognosis and a negative correlation with an average expression level of a proliferation gene for prediction of breast cancer prognosis.
For reference, the above-mentioned nucleotide and protein work can be referred to the following references (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al Inc., San Diego, Calif. (1990), < RTI ID = 0.0 > Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press De Andres et al., BioTechniques 18: 42044 (1995); Held et al., ≪ Diagnostic 2: 84-91 (2000); K. Specht et al., Am. J. Pathol., 158: 419 -29 (2001)).
Therefore, the system of the present invention can be used as an automated process for predicting and diagnosing the prognosis of breast cancer patients, and thus it can be used for the purpose of judging the necessity of chemotherapy and suggesting clues about the direction of breast cancer treatment in the future.
Figure 1 shows a schematic diagram of the method of the invention.
Figure 2 is a flow chart of the RNA isolation procedure in the FFPE sample of the method of the present invention.
FIG. 3 shows the results of analysis of 9 breast cancer prognostic amplification genes (P-genes), 6 breast cancer prognostic immune genes (I-genes), 5 standard genes (O-genes) (R-gene) of breast cancer. In the figure, the red display area is the area where the individual Cp value of each gene is measured, the green display area is not expressed, the Cp value has a very high Cp value, or the inflection point occurs. Is an unmeasured area.
Fig. 4 shows an example of a multiplex Cp measurement result.
Hereinafter, the present invention will be described in detail with reference to examples.
However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
≪ Example 1 >
FFPE
In the sample
RNA
Separation of
FFPE - treated breast cancer patients were used as samples. The FFPE sample was cut into a thickness of 5 to 10 μm using a rotary microtome and mixed with a buffer for FFPE (FFPE buffer, VERSANT tissue preparation reagents, Box 1, siemens), incubated at 80 ° C. for 30 minutes, Respectively. The temperature was lowered to 65 ° C, proteinase K (VERSANT tissue preparation reagents, Box 2, siemens) was mixed and incubated for 30 minutes. Magnetic beads (VERSANT tissue preparation reagents, Box 1, siemens) were mixed and incubated at 65 ° C for 15 minutes to allow cell debris to adhere, and magnetic force was applied to the bottom of the tube, And the upper solution was transferred to a new tube containing magnetic beads and lysis buffer (VERSANT tissue preparation reagents, Box 1, siemens) in advance. At this time, the paraffin layer formed on the upper part of the tube is not transferred to the new tube.
And incubated at room temperature for 10 minutes to bind the nucleic acid molecule to the magnetic beads. The magnetic beads were separated by applying a magnetic force to the bottom of the tube, and the supernatant was removed and then washed with washing buffer 1, 2, 3, VERSANT tissue preparation reagents, Box 1, siemens. The elution buffer (VERSANT tissue preparation reagents, Box 1, siemens) was added thereto, followed by incubation at 70 ° C for 10 minutes to elute nucleic acid molecules. The supernatant from which the nucleic acid molecules were eluted was separately transferred to a new tube to remove the magnetic beads, followed by DNase I (VERSANT tissue preparation reagents, Box 3, siemens) at 37 ° C for 10 minutes to remove the DNA.
≪ Example 2 >
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The RNA isolated in Example 1 was mixed with RT-qPCR premix (quantifast multiplex RT-PCR kit, qiagen). The RT-qPCR premix contains a reverse transcriptase for cDNA synthesis, a DNA polymerase for PCR, a fluorescent dye, and a buffer for RT-PCR. PCR amplification was carried out at 95 ° C for 15 seconds, at 95 ° C for 15 seconds, and at 60 ° C for 30 seconds. The RT-qPCR process was performed with a cycle of 40 ° C. for 30 seconds, and Cp (Crossing point, Ct) values were measured for each gene (LightCycle 480, Roche)
As a result, the Cp value for gene P1 was 30.49, the Cp value for gene P2 was 27.03, the Cp value for gene I1 was 26.25, and the Cp value for gene I2 was 25.83. In addition, the Cp values for standard genes 1, 2 and 3 were 23.63, 22.56 and 24.60, respectively.
< Example 3>
Cp
Estimates of breast cancer prognosis using value
It is necessary to normalize the calculated Cp value since it is a reflection of an individual's expression deviation. For the standardization, the average Cp value of the standard gene was calculated as 23.58 by the geometric mean calculation method. Standardized expression levels for each of the target genes, the p-gene and the i-gene (i-gene)
Standardized expression level = 2 - ( target gene Cp value) / 2 - (standard gene average Cp value)
Respectively. Expression levels of the P1, P2, I1 and I2 target genes in Example 2 were 0.008, 0.091, 0.157 and 0.210, respectively. In this case, the I2 gene expresses 26.25 times (0.210 / 0.008) It can be said that it is a lot.
Using survival data from the data set and the selected p-gene and i-gene, AFT prognosis prediction models for metastases in early breast cancer patients were generated. Using the survival information of the Discovery data set, a generational life table for each year was created to calculate the approximate risk.
The probability of death from household life table is unimodal, so it is predicted that weave, logarithmic logistic, and lognormal distribution are appropriate. The covariates to be included in the prognostic prediction model are p.mean and i.mean. If p.mean is the average value of p-genes, i.mean is the average value of i-genes.
As a result of applying the Weibull, logarithmic logistic, and logarithmic normal distribution to three models, it is most suitable for the lognormal distribution. Akaike's information criterion (AIC) was used to select the final model according to the lognormal distribution. The final estimated model for a frozen sample corresponding to the present invention instead of the FFPE sample is as follows.
log (T) = -0.689 x p.mean + 0.274 x i.mean + 3.219
According to the estimated model, the proliferation has a negative correlation (-0.689, p value = 2.47 x e-17) with the survival time (T) do. Conversely, i.mean has a positive correlation with survival time (0.274, p value = 3.69 x e-11), which means that the longer the immune response is, the longer the survival time is. Analysis of the above variables suggests that proliferation plays a decisive role in the prognosis of breast cancer, and the more prominent it becomes, the worse the prognosis, while the immune response acts as a defense mechanism against the rapid proliferation.
As described above, since the system of the present invention can predict and diagnose the prognosis of patients with breast cancer through an automated process, it can be usefully used for judging the necessity of chemotherapy and suggesting clues about the direction of breast cancer treatment in the future.
Claims (12)
(a) treating a formalin-fixed paraffin-embedded (FFPE) sample containing a patient's breast cancer tissue with a buffer for paraffin removal and cell lysis;
(b) treating the sample of step (a) with proteinase K;
(c) removing protein or cell debris from the sample of step (b);
(d) treating the sample of step (c) with DNAase I (DNase I) to obtain RNA;
(e) mixing the RNA with a primer set capable of amplifying the detection gene and an RT-qPCR premix;
(f) generating cDNA (complementary DNA) in the mixture of step (e);
(g) performing a PCR reaction using the cDNA as a template and using the primer set as a PCR primer;
(h) measuring the Cp value for the PCR reaction;
(i) Cp values for proliferative gene for breast cancer prognosis prediction diagnosis and Cp value for immune gene predictive of breast cancer prognosis prediction are shown as CTBP1, TATA-binding protein (TBP), hydroxymethylbilane synthase (HMBS) Standardized expression level by standardizing the Cp values of two or more selected standard genes in a group consisting of CUL1 (cullin 1) and UBQLN1 (Ubiquilin-1) to a geomean;
(j) Standardized breast cancer prognosis prediction The lower the standardized expression level of the proliferation gene for diagnosis and the higher the normalized expression level of the immune gene for the prediction breast cancer prognosis prediction diagnosis, the better the prognosis for breast cancer.
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| Applied Biosystems 社의 카탈로그 "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR" (2004)* |
| Bernard, PS., et al., Clinical Chemistry Vol.48(8), pp.1178-1185 (2002)* |
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