A method and kit for detecting cancer-related genes using amplified competitive sequences.

Abstract

The present invention provides a method and composition for a simple PCR-based means for generating a risk score based on a gene signature for the prognosis of breast cancer recurrence and for predicting treatment effectiveness. The present invention uses competitive amplification to encode the gene signature into oligonucleotide design, enabling five downstream amplifications and subsequent analysis directly and making it suitable for on-site implementation.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of in vitro diagnostics. BACKGROUND ART

[0002] Various tests have been developed to determine different parameters for a specific tumor in an individual. For example, specific biomarkers such as overexpression or underexpression of a particular gene in a tumor specimen can be used to diagnose the presence of cancer or provide an indicator for conditions such as metastasis.

[0003] One specific well-known test, the Oncotype DX test from Exact Sciences, was developed to determine the likelihood of breast cancer recurrence (i.e., prognosis) and the potential benefit of chemotherapy treatment (i.e., prediction) in patients with specific types of cancer.

[0004] Such tests are valuable because they provide guidance on treatment options to patients who are most likely to benefit, while allowing patients who are less likely to benefit to avoid them, helping to spare these patients from unnecessary side effects of ineffective treatments. For these latter patients, a different treatment may be selected instead. Where cancer recurrence is more likely to occur, specific screening programs can be implemented.

[0005] The Oncotype DX test is a well-known 21-gene test that includes real-time reverse transcription PCR (real-time rt-PCR), which includes a set of primers and probes that can be used to initially quantify the concentrations of 21 genes in a patient sample (each in a separate reaction). For example, Kalinsky K et al.2021 et al.,New England Journal of Medicine,2021,385(25):2336-2347.doi:10.1056 / NEJMoa2108873;Paik S et al,New England Journal of Medicine,2004,351(27):2817-2826.doi:10.1056 / NEJMoa041588;Sparano JA et al,2018 New England Journal of Medicine.2018,379(2):111-121.doi:10.1056 / NEJMoa1804710;and Cronin M et al.,Clinical See Chemistry, 2007.53(6):1084-1091.doi:10.1373 / clinchem.2006.076497.

[0006] The raw CT values ​​of each gene are converted into a risk score via an algebraic "algorithm" to determine the patient's risk of recurrence. The outcome of the trial is a single value called the "recurrence score (RS score)," which is used in combination with other characteristics such as the patient's age, lymph node metastasis positive / negative status, and HER2 status to provide prognostic and predictive outcomes.

[0007] This test will be conducted in a single, centralized facility using a custom-designed high-throughput protocol.

[0008] There is a need to provide a simple means of achieving an RS score that can be interpreted in the same way as the Oncotype DX RS score, which fits the current clinical pathway, and can be done in a decentralized manner. For example, the test could be performed at the same hospital where the patient is being treated, resulting in a faster and less stressful process for the patient.

[0009] The present invention aims to solve this problem. [Overview of the Initiative]

[0010] The present invention provides a simplified means for generating RS scores by utilizing a competitive amplification network (CAN), which is described in International Publication 2022 / 074392(A1) (in its entirety incorporated herein) and Goertz et al., "Competitive Amplification Networks enable molecular pattern recognition with PCR," biorxiv, 2023, doi:10.1101 / 2023.06.29.546934, and is available at https: / / www.biorxiv.org / content / 10.1101 / 2023.06.29.546934v1.full.pdf (each of which is in its entirety incorporated herein by reference).

[0011] CAN enables the collectively interpreted expression levels of multiple genes in a single reaction. Rather than quantifying each RNA transcript individually, either the quantity or raw CT is input into the interpretation algorithm, which is how current Oncotype DX tests work, but CAN incorporates algorithmic interpretation into the molecular design of the components.

[0012] Rather than using different fluorescence channels to indicate the absolute amounts of different components, CAN uses fluorescence color to encode the relative concentration of each target compared to a pre-discrimination boundary. Different boundary concentrations, determined empirically, are used for each target gene to best separate “disease-negative” (low-risk) patients from “disease-positive” (high-risk) patients. Numerous RNA transcripts are analyzed in a single reaction (or in multiple modules of individual reactions), each contributing to the same two fluorescence channels. The relative intensities of the two channels at the end of the reaction can be interpreted as an indicator of whether the target gene is more likely to indicate that the patient is at low or high risk. An exemplary representation of CAN can be seen in Figure 2.

[0013] The test of the present invention, sometimes referred to as the OncoSignatur Breast test, is a molecular pathology assay for determining the risk of breast cancer recurrence after surgery in women. This test analyzes the expression of specific genes (the same genes used in the Oncotype DX test) in the patient's excised tumor and converts the concentration of each gene transcript into a score (RS score) representing the likelihood of recurrence.

[0014] CAN modifies the design of RT-qPCR systems by incorporating long-chain synthetic oligonucleotides called competitors, and by a novel method for selecting primer and probe sequences and determining their concentrations in the kit. One or more competitor oligos are included, each corresponding to one of the natural RNA targets. A given competitor concentration is related to the pre-discrimination boundary of each RNA target. Different probes may be designed specifically for each competitor and each target sequence, or there may be two probes (one designed to be specific to the competitor and the other to be specific to the target).

[0015] During enzymatic RT-qPCR amplification, these competitors compete with the native target or other competitors for the primer group. The amplification rate of a given amplicon (and therefore the growth of each fluorescence signal) slows down as its corresponding primer group is consumed, and stops completely when the primer group is exhausted. This competition for the primer group ensures that the final fluorescence intensity of the signals derived from the native RNA and / or synthetic competitors reflects the relative concentrations of the two at the start of the reaction.

[0016] The final fluorescence intensity can be correlated with empirical patient data through one of several methods known in the field.

[0017] This invention makes it possible to perform this evaluation in an environment close to the patient, such as a molecular pathology laboratory in the same hospital where the surgery was performed. This is in contrast to existing tests such as Oncotype DX, which require patient samples to be sent to Exact Sciences facilities in California.

[0018] This invention is easier to use than related tools of the prior art. Only 1 to 6 aliquots of purified RNA from a processed patient tumor sample are required to perform the test, significantly fewer than other kits that require 12 to 63 or more. This allows for rapid testing while reducing variability resulting from splitting the sample across many reactions, thereby improving performance.

[0019] In some embodiments, the methods and kits of the present invention, for example, the methods and kits referred herein to as the OncoSignatur Breast Test (OSB) Method and Kit, may be kits that can be readily performed by molecular pathology technicians on readily available polymerase chain reaction (PCR) equipment (i.e., real-time thermocyclers). Data from the equipment can be correlated with empirical risk through any of several means known in the art. In some embodiments, the interpretation of the RS score derived by the Method and Kit is the same as the interpretation of the RS score derived from the Oncotype DX test.

[0020] In some embodiments, the kit includes a mixture of many synthetic oligonucleotides (oligonucleotides) along with a mixture of enzymes and other reagents. The synthetic oligonucleotides are the subject of the present invention and any of several reagent mixtures well known to those skilled in the art can be used.

[0021] The present invention uses a concept similar to that of reverse transcription quantitative PCR mixture (RT-qPCR), which functions as follows: The reagent mixture first converts a specific native gene expression product (RNA transcript) in the patient sample into complementary DNA (cDNA), and then, with the help of primer oligos, generates copies of the cDNA sequence. This process is known as amplification, and these copies are known as amplicons. Amplification occurs via the extension of primer oligos, which are consumed in the process. Furthermore, probe oligos specific to each native target are degraded by enzymes in the reagent mixture. These probes are synthesized using a fluorescent molecule bound to one end and a fluorescent quenching molecule bound to the other end. By degrading the probes, these two labels are separated, the quenching of the fluorophores is removed, and a fluorescent signal is generated. Typically, this fluorescent signal is monitored until it exceeds a certain threshold known as CT, and the number of thermal cycles required to exceed this threshold is quantitatively related to the initial concentration of the relevant RNA sequence. When multiple target genes are present, their RNA transcripts are quantified individually. This is done by using probes bound to fluorescent molecules, allowing each transcript to be measured either in the same reaction or by using physically separate reactions.

[0022] The Oncotype DX test uses the CT value to reach the RS value. This invention uses the endpoint fluorescence intensity achieved after a specific number of cycles to induce the RS value.

[0023] Amplification of 21 genes and subsequent fluorescence detection performed by the Oncotype DX test requires 42 primer sets and 21 probes. These 42 primer sets and 21 probes cannot be simply combined into a single reaction with the expectation of equivalent (or even acceptable) performance, nor can existing high-throughput protocols be simply adopted to miniaturize them for clinical laboratories.

[0024] Furthermore, when using CAN in accordance with the present invention, the algorithm must be "embedded" in the design of the reaction components. In Oncotype DX, the raw CT value of each target is supplied to an algebraic equation to determine a patient's recurrence risk, whereas in the present invention, competitive amplification behavior must be carefully adjusted to generate an appropriate signal that directly indicates the recurrence risk. This likewise requires a combination of skilled knowledge, custom computer modeling, careful experimental design, and trial and error.

[0025] The reaction components described herein, such as primer sets and competitive oligonucleotides, already have the algorithm "embedded", and determining risk using the primer set / competitor set described herein is a simple step of performing 1 to 5 separate PCR amplification reactions, reading endpoint fluorescence, and using it for a simple calculation. One form of the above calculation is shown in Figure 16.

[0026] Although CAN is known, in addition to the above difficulties in designing any new primer set, i.e., which compounds increase as the size of the primer set increases, the design of competitors and component concentrations must be specific to each new application and gene set.

[0027] The present invention provides specifically designed specific reaction components (probes and primer sets and competitive oligonucleotides) and conditions for multiplex amplification, and for transmitting appropriate algorithmic information to endpoint fluorescence. [Mode for Carrying Out the Invention]

[0028] The present invention is as described in the claims.

[0029] The Oncotype DX assay analyzes the expression levels of 21 genes - 5 housekeeping genes and 16 highly informative genes. These genes are grouped together into 5 distinct modules, as described below. Prior art assays require analysis of the expression level of each gene and determination of CT values using RT-PCR.

[0030] The assay of the present invention does not determine the expression levels of individual genes. Instead, the present invention provides an endpoint fluorescence readout after a predetermined number of amplification cycles for genes within a given module. Further, the endpoint fluorescence of a module does not merely provide readout information of the amount of starting material (i.e., the expression level of a particular gene in the sample); rather, the readout information incorporates, for example, information on the relevance of each particular marker in a gene signature that predicts breast cancer recurrence. This information is encoded into the amplification reaction by the specific design of competing oligonucleotides corresponding to each gene in the panel.

[0031] The output of the amplification reaction is the endpoint fluorescence level for each module, which can be converted via a simple algorithm to an RS score (the same RS score as obtained via the Oncotype DX assay). The data in the Examples show that the RS scores obtained by the present invention correlate with the Oncotype DX RS scores obtained by performing the Oncotype DX algorithm on synthetic samples.

[0032] For example, for the case where 5 different modules are amplified separately, such that each module has its own endpoint fluorescence value, one form in which the RS score (or OSB score) can be calculated is as follows.

[0033] [Table 1]

[0034] "Prol" refers to the endpoint signal from the Prol module for a given sample, divided by the signal from its respective control reaction (a reaction with a competitor, primer group, or probe only, which shows the maximum possible signal from the Prol module). Estp, Invp, Her2, and Hskp are defined similarly.

[0035] The form of the above formula is considered suitable for use with whatever set of primers / competitors / probes is used for a given module (i.e., any of two or three different sets of primers / competitors / probes described herein for amplification of each module), although the specific numbers will vary, and those skilled in the art will be able to easily determine what those numbers should be.

[0036] Here, the method includes all of the following: Amplification of the housekeeping module using the following oligonucleotides: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with Housekeeping Fluorescent Label. and The infiltration module is amplified using the following oligonucleotides: The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; and The amplification of the proliferation module is performed using the following oligonucleotides: The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; and The Her2 module is amplified using the following oligonucleotides: The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; and The estrogen module is amplified using the following oligonucleotides: The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and Universal estrogen module probe oligonucleotides, In this case, the numbers may be as follows:

[0037] [Table 2]

[0038] Accordingly, the present invention provides methods for amplifying target polynucleotides. These methods can be used to obtain outputs that can be used in algorithms for generating risk scores or RS scores comparable to the RS score of an oncotype DX test. In this specification, the RS score determined by the present invention is referred to as the OSB score.

[0039] The present invention provides various methods and compositions for amplifying target nucleic acids, methods for obtaining scores that can be used to predict breast cancer recurrence, and methods for determining the likelihood of treatment being effective; as well as methods for prognosis diagnosis and determining the likelihood of treatment being effective.

[0040] Therefore, in one embodiment, the present invention is a method for amplifying at least two, i.e., multiple, target polynucleotides in a sample, the following: a) A step of providing a sample potentially containing at least two, i.e., multiple, target polynucleotides, b) Providing at least two, i.e., multiple sets of target competitive amplification oligonucleotides, wherein each set of target competitive amplification oligonucleotides is designed to amplify a particular target polynucleotide, and each target to be amplified has a corresponding set of target competitive amplification oligonucleotides within each set: i) Competitive polynucleotides; ii) A first primer and a second primer that can hybridize to a specific target polynucleotide and a first competing polynucleotide of a set, wherein hybridization of the first primer and the second primer to the specific target polynucleotide enables amplification of a portion of the specific target polynucleotide that generates a target amplicon, and hybridization of the first primer and the second primer to the first competing polynucleotide enables amplification of the first competing polynucleotide or a portion of the first competing polynucleotide that generates a competing amplicon; iii) A probe oligonucleotide comprising a fluorescent label and capable of hybridizing to a first competing amplicon or a first target amplicon, The first competing polynucleotide in each set is designed to have a different amplification kinetics than that of the first target polynucleotide in each set, step, c) A step in which the primer extension reaction is initiated, At least two, i.e., multiple, portions of the target polynucleotides are amplified, resulting in the generation of at least two, i.e., multiple different target amplicons, corresponding to each specific target; Amplification occurs in each competitive polynucleotide or a portion of each competitive polynucleotide in each set of target competitive amplification oligonucleotides, resulting in the generation of at least two, i.e., multiple, different competitive amplicons. At least two, i.e., multiple, target polynucleotides are cDNA molecules derived from the first and second, or multiple, different mRNA polynucleotides present in the sample obtained from the subject. The present invention provides a method comprising the step of expressing at least first and second, i.e., multiple mRNA polynucleotides, from the following genes: CD68, CTSV, MMP11, AURKA, BIRC5, CCNB1, MKI67, MYBL2, ERBB2, GRB7, BAG1, BCL2, ESR1, GSTM1, PGR, SCUBE2, ACTB, GAPDH, GUSB, RPLP0, TFRC or a group consisting of these.

[0041] In some embodiments, a) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from CD68, the corresponding set of target competitive amplification oligonucleotides includes a CD68 competitive polynucleotide; a CD68 first primer; a CD68 second primer; and a CD68 probe oligonucleotide; b) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from CTSV, the corresponding set of target competitive amplification oligonucleotides comprises: CTSV competitive polynucleotide; CTSV first primer; CTSV second primer; and CTSV probe oligonucleotide; c) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from MMP11, the corresponding set of target competitive amplification oligonucleotides includes: MMP11 competitive polynucleotide; MMP11 first primer; MMP11 second primer; and MMP11 probe oligonucleotide; d) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from AURKA, the corresponding set of target competitive amplification oligonucleotides includes the AURKA competitive polynucleotide; the AURKA first primer; the AURKA second primer; and the AURKA probe oligonucleotide; e) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from BIRC5, the corresponding set of target competitive amplification oligonucleotides includes a BIRC5 competitive polynucleotide; a BIRC5 first primer; a BIRC5 second primer; and a BIRC5 probe oligonucleotide; f) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from CCNB1, the corresponding set of target competitive amplification oligonucleotides includes the CCNB1 competitive polynucleotide; a CCNB1 first primer; a CCNB1 second primer; and a CCNB1 probe oligonucleotide; g) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from MKI67, the corresponding set of target competitive amplification oligonucleotides comprises: MKI67 competitive polynucleotide; MKI67 first primer; MKI67 second primer; and MKI67 probe oligonucleotide; h) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from MYBL2, the corresponding set of target competitive amplification oligonucleotides comprises the MYBL2 competitive polynucleotide; the MYBL2 first primer; the MYBL2 second primer; and the MYBL2 probe oligonucleotide; i) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from ERBB2, the corresponding set of target competitive amplification oligonucleotides includes: ERBB2 competitive polynucleotide; ERBB2 first primer; ERBB2 second primer; and ERBB2 probe oligonucleotide; j) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from GRB7, the corresponding set of target competitive amplification oligonucleotides includes the GRB7 competitive polynucleotide; a primer for GRB7 first; a primer for GRB7 second; and a GRB7 probe oligonucleotide; k) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from BAG1, the corresponding set of target competitive amplification oligonucleotides includes the BAG1 competitive polynucleotide; the BAG1 first primer; the BAG1 second primer; and the BAG1 probe oligonucleotide; l) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from BCL2, the corresponding set of target competitive amplification oligonucleotides includes a BCL2 competitive polynucleotide; a BCL2 first primer; a BCL2 second primer; and a BCL2 probe oligonucleotide; m) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from ESR1, the corresponding set of target competitive amplification oligonucleotides includes: ESR1 competitive polynucleotide; ESR1 first primer; ESR1 second primer; and ESR1 probe oligonucleotide; n) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from GSTM1, the corresponding set of target competitive amplification oligonucleotides includes the GSTM1 competitive polynucleotide; the GSTM1 first primer; the GSTM1 second primer; and the GSTM1 probe oligonucleotide; o) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from PGR, the corresponding set of target competitive amplification oligonucleotides includes: PGR competitive polynucleotide; PGR first primer; PGR second primer; and PGR probe oligonucleotide; p) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from SCUBE2, the corresponding set of target competitive amplification oligonucleotides includes the SCUBE2 competitive polynucleotide; the SCUBE2 first primer; the SCUBE2 second primer; and the SCUBE2 probe oligonucleotide; q) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from ACTB, the corresponding set of target competitive amplification oligonucleotides includes the ACTB competitive polynucleotide; the ACTB first primer; the ACTB second primer; and the ACTB probe oligonucleotide; r) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from GAPDH, the corresponding set of target competitive amplification oligonucleotides includes the GAPDH competitive polynucleotide; the GAPDH first primer; the GAPDH second primer; and the GAPDH probe oligonucleotide; s) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from GUSB, the corresponding set of target competitive amplification oligonucleotides is: GUSB competitive polynucleotide; GUSB first primer; GUSB second primer; and GUSB probe oligonucleotide; t) If at least two, i.e., one of multiple mRNA polynucleotides, is expressed from RPLP0, the corresponding set of target competitive amplification oligonucleotides comprises: RPLP0 competitive polynucleotide; RPLP0 first primer; RPLP0 second primer; and RPLP0 probe oligonucleotide; and / or w) If at least two, i.e., one of multiple mRNA polynucleotides, are expressed from the TFRC, the corresponding set of target competitive amplification oligonucleotides includes the TFRC competitive polynucleotide; the TFRC first primer; the TFRC second primer; and the TFRC probe oligonucleotide.

[0042] In some embodiments, a single probe can be used to detect multiple target polynucleotides. Therefore, in one embodiment, the sequence of a probe oligonucleotide for a given set of competing oligonucleotides may be the same as the sequence of a probe oligonucleotide for a different set of competing oligonucleotides. In some embodiments, the fluorescent label of a probe oligonucleotide for a given set of competing oligonucleotides may be the same as the fluorescent label of a probe oligonucleotide for a second, different set of competing oligonucleotides.

[0043] In some embodiments, multiple target polynucleotides are grouped into a housekeeping (HK) module, an invasion module, and / or an invasion module; a proliferation module; a HER2 module; and an estrogen module. a) The housekeeping module includes or consists of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: ACTB, GADPH, GUSB, RPLP0, and TFRC; b) The infiltration module contains or consists of a target polynucleotide, which is a cDNA molecule derived from mRNA polynucleotides expressed from the following genes: CD68, CTSV, and MMP11; c) The proliferation module contains or consists of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: AURKA, BIRC5, CCNB1, MKI67, and MYBL2; d) The HER2 module contains or consists of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: ERBB2 and GRB7 or the group consisting of them; and / or e) The estrogen module contains or consists of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: BAG1, BLC2, ESR1, GSTM1, PGR, and SCUBE2, or a group consisting of these genes.

[0044] Any number of target polynucleotides can be amplified within a given module; however, preferably, each target polynucleotide is amplified. Therefore, in one embodiment, the method includes amplified each target within one module.

[0045] Target polynucleotides of any number of modules can be amplified. In some embodiments, the method includes amplified target polynucleotides of a housekeeping module, an invasion module, a proliferation module, a HER2 module, and / or an estrogen module. The housekeeping module, invasion module, proliferation module, HER2 module, and / or estrogen module are amplified in separate reactions, for example, in separate wells of a reaction plate, or in the same reaction, i.e., within the same well of a reaction plate.

[0046] In some embodiments, each probe within the module is labeled with the same fluorescent label, which is the fluorescent label for the module. In some configurations of this embodiment, The fluorescent module label for each target-related probe in the housekeeping module is labeled with the fluorescent label for the housekeeping module; The module-specific fluorescent label for each probe related to the target in the invasion module is labeled with the invasion module-specific fluorescent label; The module-specific fluorescent label for each target-related probe in the proliferation module is labeled with the proliferation module-specific fluorescent label; The module-specific fluorescent label for each target-related probe in the HER2 module is labeled with the HER2 module-specific fluorescent label; Each target-related probe in the estrogen module is labeled with the module's fluorescent label.

[0047] As can be understood, target polynucleotides of multiple modules can be amplified in one (i.e., the same) amplification reaction. That is, multiple modules can be amplified in a multiplexed reaction. Therefore, in some embodiments, the method includes amplifying target polynucleotides from two, three, four, or five modules in the same amplification reaction.

[0048] The terms "fluorescent label for housekeeping module," "fluorescent label for proliferation module," "fluorescent label for invasion module," "fluorescent label for HER2 module," and "fluorescent label for estrogen module" include the meaning of any fluorescent label. As described elsewhere in this specification, the same fluorescent label is used within a given module, and therefore, a reference to a fluorescent label for housekeeping module is intended to refer to a fluorescent label present on a probe associated with the housekeeping module.

[0049] In some embodiments, the fluorescent labels for each module have different, interchangeable fluorescent labels. For example, if the housekeeping module is amplified in the same amplification reaction as the HER2 module and the estrogen module, the fluorescent labels for the housekeeping module, the HER2 module, and the estrogen module must be distinguishable from each other, i.e., interchangeable. Those skilled in the art are well aware of the concept of multiplexing and the requirements for selecting appropriate probe labels that do not interfere with each other, are readable, and provide output signals that can be distinguished from the output signals of other probes.

[0050] A key part of the present invention requires that each group of target polynucleotides in a module be amplified together using a probe having the same fluorophore. Multiplexed probes for PCR amplification are well known, and those skilled in the art will recognize that appropriate fluorescent labels can be selected so that multiple modules can be analyzed together in the same amplification reaction using probes with compatible fluorophores. Exemplary compatible fluorophores can be selected, for example, from Figure 1. In some embodiments, one or more modules are then run together in the same amplification reaction, and each module uses a probe having a different but compatible fluorescent label. In other embodiments, each module is analyzed separately, which means that different fluorescent labels can be used, but it is also possible to use the same fluorescent label. Figure 5 shows some different variations of this. Different types of shading indicate different fluorescent labels. For example, Figure 5A shows five separate amplification reactions, each using probes labeled with the same fluorescent label. Since the amplification reactions are independent, there is no need to consider the compatibility of the fluorescent labels. Figure 5B again shows five independent amplification reactions, but in this case the probes are labeled with different fluorescent labels. Figure 5C shows two different amplification reactions. One reaction amplifies and analyzes four modules—in this reaction, the probe associated with each module is labeled with a different, compatible fluorescent label (indicated by different shading). A second reaction amplifies a single module. The probe associated with this module may be labeled with a different fluorescent label than the one used in the first reaction, or it may be the same as one of the labels used in the first reaction. Since this second reaction proceeds independently of the first reaction, the choice of fluorescent label is largely irrelevant (depending on whether nonspecific fluorophores are present during the reaction). Figure 5D shows the case where only four of the modules are amplified, again using a probe with a different fluorescent label specific to each module. Figure 5E shows the same setup as in Figure 5A, but in the presence of the nonspecific marker dye EvaGreen.Figure 5F shows the same configuration as Figure 5B, but with some modules amplified in the presence of the EvaGreen dye and some modules amplified in the presence of the EvaRuby dye. Figures 5G and 5H again show the same configuration as Figures 5C and D in the presence of different nonspecific fluorescent markers. It will be apparent to those skilled in the art that there are many different configurations in which the method of the present invention can be carried out. A key feature is that the endpoint fluorescence from each module must be determinable, whether by performing five distinct individual amplification reactions (one for each module), or by a single reaction using five different compatible fluorescent markers, and by all combinations therein. Those skilled in the art will know how to select compatible fluorophores and labels, for example, as described below.

[0051] Fluorescent labels The use of multiplex PCR with different fluorescently labeled probes is commonplace in the art, and those skilled in the art can adequately select appropriate fluorescent labels for labeling each probe oligonucleotide in a set of competing amplification oligonucleotides. For example, those skilled in the art will understand that when selecting appropriate fluorescent labels for multiplex PCR or multiplex qPCR assays, several considerations should be taken into account to ensure accurate and reliable results.

[0052] Unique Reporter Dyes: Each different fluorescent label used in the amplification reaction of the set of the present invention must have a unique reporter dye with a different spectrum. This makes it possible to distinguish amplifications based on the fluorescence signal.

[0053] Crosstalk Reduction: Crosstalk, which is interference of fluorescence signals from one reporter to an adjacent channel, must be avoided. Select a set of dyes with emission spectra that are sufficiently far apart from each other so that they can be easily resolved. The selection of optical specifications should match the thermal cycler used, taking into account the excitation light source (laser, lamp, or LED) and the detection filter.

[0054] Compatibility: The selected fluorescent label should be compatible with the instrument being used. This means there should be no significant overlap between the excitation and emission spectra of the fluorophore and the detection channel of the instrument. Crosstalk can lead to false-positive amplification and interfere with quantification.

[0055] Calibration: Real-time qPCR instruments must be calibrated for a specific set of dyes selected for the experiment. Calibration ensures that the instrument can accurately detect the emission spectrum for each dye and minimize background and signal overlap.

[0056] Dye Selection: Those skilled in the art can select dyes compatible with a particular instrument model. Tools such as the PrimeTime Multiplex Dye Selection Tool can help identify dyes compatible with various instrument models.

[0057] Some dyes are compatible with the instrument, but the instrument may require calibration, so the instrument needs to be calibrated. In such cases, an alternative set of dyes may be available. Fluorescent labels should be selected to have appropriate excitation wavelengths and to have no or minimal overlap in their emission spectra. Those skilled in the art can consider the total fluorescence intensity.

[0058] In some cases, any of the amplification reactions of the present invention may include a further dye or label used as a passive reference label. For example, in some examples, the amplification reaction includes a fluorescent dye that intercalates to the polynucleotide DNA in the amplification reaction, or otherwise binds nonspecifically. There are many nonspecific dyes that can be used for this purpose, e.g., Sybr Green dye, EvaGreen dye, or EvaRuby dye. Furthermore, other reference dyes such as ROX may be included in the amplification reaction. The selection of fluorescent labels for use with the probe oligonucleotides of the present invention should also take into account the spectra of these nonspecific non-probe fluorescent labels and dyes.

[0059] Minimizing Crosstalk: To reduce crosstalk in multiple reactions, those skilled in the art know to select probes with low background fluorescence. Quenchers can also be used. Therefore, in any of the methods and compositions of the present invention, any probe oligonucleotide may include a quencher in addition to a fluorescent label. Efficient dark quenchers can significantly reduce background fluorescence, especially when used in combination with a secondary internal quencher. Dual-quench probes offer advantages such as clear endpoint signals and faster quantitative cycle (Cq) values. Consistency in the type of quencher used (all dark quenchers or all fluorescent quenchers) is recommended for multiplexed assays to minimize background fluorescence.

[0060] Therefore, those skilled in the art can easily select suitable fluorescent labels for use with any of the probe oligonucleotides of the present invention.

[0061] In some embodiments, the sequence of probe oligonucleotides for a given set of competing oligonucleotides may be the same sequence of probe oligonucleotides for different sets of competing oligonucleotides.

[0062] In some embodiments, one or more modules include a single probe oligonucleotide, which can optionally hybridize to a competing amplicon or a target amplicon. The probes described herein hybridize to a competing amplicon, but can also be designed to hybridize to a target amplicon.

[0063] In some embodiments, multiple probes, i.e., at least two or more probes, are used to obtain fluorescence readout of each target polynucleotide of a given module. This is sometimes called the “multiprobe” method. In the multiprobe method, one or more sets of competitive amplification oligonucleotides used to amplify the target polynucleotide of a given module include one or more probe oligonucleotides having a different sequence from one or more other probe oligonucleotides in the same set of competitive amplification oligonucleotides used for a given module, i.e., the module targets are amplified using the same universal module probes designed to hybridize to the competitive amplicons and / or target amplicons of each target of the module, with different probe oligonucleotides having nucleic acid sequences different from one or more other probes in the set of competitive amplification oligonucleotides. Table 1 shows competitives and probe sequences suitable for the multiprobe method.

[0064] In other embodiments, a single fluorescently labeled probe is used to provide a fluorescent readout for each target polynucleotide of a given module; that is, a single probe can hybridize to each of the competing amplicons generated for targets produced from genes including, for example, BAG1, BCL2, ESR1, GSTM1, PGR, and SCUBE2 genes, i.e., genes containing the estrogen module. This is sometimes referred to as the “single probe” method. In these embodiments, each set of competitive amplification oligonucleotides used to amplify the target polynucleotides of a given module all contain the same probe oligonucleotide sequence; that is, the module's targets are amplified using a set of competitive amplification oligonucleotides, each containing the same universal module probe designed to hybridize to the competing amplicons and / or target amplicons of each target of the module. Table 2 shows competitive agents and probe sequences suitable for the single probe method.

[0065] In some embodiments, a target polynucleotide of a single module is amplified in the amplification reaction in the absence of amplification of a target polynucleotide of another module.

[0066] In some embodiments, the module probe is any fluorescent label, independent of the selection of the fluorescent label used in the amplification of the target polynucleotide of other modules in other amplification reactions.

[0067] In some embodiments, amplification is performed as follows: First amplification reaction for amplification of the infiltration module; Second amplification reaction for amplification of the growth module; Third amplification reaction for amplification of the HER2 module; The fourth amplification reaction for the amplification of the estrogen module; It includes five different amplification reactions, including a first amplification reaction for amplification of the housekeeping module. The selection of fluorescent labels for each module is independent.

[0068] As can be understood, if each module is amplified in a separate amplification reaction, there is no need to distinguish between the amplification of different modules. Therefore, if each module is amplified in a separate amplification reaction, the fluorophores may be incompatible between different reactions, and / or may be the same between different reactions. In some embodiments, the fluorescent labels for the invasion module, the proliferation module, the HER2 module, the estrogen module, and the housekeeping module are all the same label.

[0069] By following the disclosures provided herein, those skilled in the art can determine the sequences of competing polynucleotides, a first primer, a second primer, and a probe oligonucleotide that may be used in the methods provided herein. In preferred embodiments, the methods provided herein are carried out using the following polynucleotides: The CD68 competitive polynucleotide has the sequence of [SEQ ID NO: 20], [SEQ ID NO: 81], or [SEQ ID NO: 110]; the first CD68 primer has the sequence of [SEQ ID NO: 17]; the second CD68 primer has the sequence of [SEQ ID NO: 18]; the CD68 probe oligonucleotide has the sequence of [SEQ ID NO: 19], [SEQ ID NO: 84], or [SEQ ID NO: 108]; the CTSV competitive polynucleotide has the sequence of [SEQ ID NO: 24], [SEQ ID NO: 82], or [SEQ ID NO: 107]; the first CTSV primer has the sequence of [SEQ ID NO: 21] The CTSV second primer has the sequence of [SEQ ID NO: 22]; the CTSV probe oligonucleotide has the sequence of [SEQ ID NO: 23] or [SEQ ID NO: 84] or [SEQ ID NO: 108]; the MMP11 competitive polynucleotide has the sequence of [SEQ ID NO: 28] or [SEQ ID NO: 83] or [SEQ ID NO: 109]; the MMP11 first primer has the sequence of [SEQ ID NO: 25]; the MMP11 second primer has the sequence of [SEQ ID NO: 26]; the MMP11 probe oligonucleotide has the sequence of [SEQ ID NO: 27] or [SEQ ID NO: 84] or [SEQ ID NO: 109] The sequence of

[108] is present; the AURKA competitive polynucleotide has the sequence of [SEQ ID NO: 32] or [SEQ ID NO: 111]; the AURKA first primer has the sequence of [SEQ ID NO: 29]; the AURKA second primer has the sequence of [SEQ ID NO: 30]; the AURKA probe oligonucleotide has the sequence of [SEQ ID NO: 31] or [SEQ ID NO: 85] or [SEQ ID NO: 112]; the BIRC5 competitive polynucleotide has the sequence of [SEQ ID NO: 36] or [SEQ ID NO: 113]; the BIRC5 first primer has the sequence of [SEQ ID NO: 33]; B The IRC5 second primer has the sequence of [SEQ ID NO: 34]; the BIRC5 probe oligonucleotide has the sequence of [SEQ ID NO: 35], [SEQ ID NO: 85], or [SEQ ID NO: 112]; the CCNB1 competitive polynucleotide has the sequence of [SEQ ID NO: 40] or [SEQ ID NO: 116]; the CCNB1 first primer has the sequence of [SEQ ID NO: 37]; the CCNB1 second primer has the sequence of [SEQ ID NO: 38]; the CCNB1 probe oligonucleotide has the sequence of [SEQ ID NO: 39], [SEQ ID NO: 85], or [SEQ ID NO: 112];The MKI67 competitive polynucleotide has the sequence of [SEQ ID NO: 44] or [SEQ ID NO: 114]; the MKI67 first primer has the sequence of [SEQ ID NO: 41]; the MKI67 second primer has the sequence of [SEQ ID NO: 42]; the MKI67 probe oligonucleotide has the sequence of [SEQ ID NO: 43] or [SEQ ID NO: 85] or [SEQ ID NO: 112]; the MYBL2 competitive polynucleotide has the sequence of [SEQ ID NO: 48] or [SEQ ID NO: 115]; the MYBL2 first primer has the sequence of [SEQ ID NO: 45]; the MYBL2 second The primer has the sequence of [SEQ ID NO: 46]; the MYBL2 probe oligonucleotide has the sequence of [SEQ ID NO: 47] or [SEQ ID NO: 85] or [SEQ ID NO: 112]; the ERBB2 competitive polynucleotide has the sequence of [SEQ ID NO: 52] or [SEQ ID NO: 117]; the ERBB2 first primer has the sequence of [SEQ ID NO: 49]; the ERBB2 second primer has the sequence of [SEQ ID NO: 50]; the ERBB2 probe oligonucleotide has the sequence of [SEQ ID NO: 51] or [SEQ ID NO: 86] or [SEQ ID NO: 118]; GR The B7 competitive polynucleotide has the sequence of [SEQ ID NO: 56] or [SEQ ID NO: 119]; the GRB7 first primer has the sequence of [SEQ ID NO: 53]; the GRB7 second primer has the sequence of [SEQ ID NO: 54]; the GRB7 probe oligonucleotide has the sequence of [SEQ ID NO: 55], [SEQ ID NO: 86], or [SEQ ID NO: 118]; the BAG1 competitive polynucleotide has the sequence of [SEQ ID NO: 60], [SEQ ID NO: 87], or [SEQ ID NO: 100]; the BAG1 first primer has the sequence of [SEQ ID NO: 57]; the BAG1 second The primer has the sequence of [SEQ ID NO: 58]; the BAG1 probe oligonucleotide has the sequence of [SEQ ID NO: 59] or [SEQ ID NO: 93] or [SEQ ID NO: 95]; the BCL2 competitive polynucleotide has the sequence of [SEQ ID NO: 64] or [SEQ ID NO: 88] or [SEQ ID NO: 97]; the first BCL2 primer has the sequence of [SEQ ID NO: 61]; the second BCL2 primer has the sequence of [SEQ ID NO: 62]; the BCL2 probe oligonucleotide has the sequence of [SEQ ID NO: 63] or [SEQ ID NO: 93] or [SEQ ID NO: 95];The ESR1 competitive polynucleotide has the sequence of [SEQ ID NO: 68], [SEQ ID NO: 89], or [SEQ ID NO: 94]; the ESR1 first primer has the sequence of [SEQ ID NO: 65]; the ESR1 second primer has the sequence of [SEQ ID NO: 66]; the ESR1 probe oligonucleotide has the sequence of [SEQ ID NO: 67], [SEQ ID NO: 93], or [SEQ ID NO: 95]; the GSTM1 competitive polynucleotide has the sequence of [SEQ ID NO: 72], [SEQ ID NO: 90], or [SEQ ID NO: 99]; the GSTM1 first primer has the sequence of [SEQ ID NO: 69]; The GSTM1 second primer has the sequence of [SEQ ID NO: 70]; the GSTM1 probe oligonucleotide has the sequence of [SEQ ID NO: 71] or [SEQ ID NO: 93] or [SEQ ID NO: 95]; the PGR competing polynucleotide has the sequence of [SEQ ID NO: 76] or [SEQ ID NO: 91] or [SEQ ID NO: 96]; the PGR first primer has the sequence of [SEQ ID NO: 73]; the PGR second primer has the sequence of [SEQ ID NO: 74]; the PGR probe oligonucleotide has the sequence of [SEQ ID NO: 75] or [SEQ ID NO: 93] or [SEQ ID NO: 95]; S The CUBE2 competitive polynucleotide has the sequence of [SEQ ID NO: 80], [SEQ ID NO: 92], or [SEQ ID NO: 98]; the SCUBE2 first primer has the sequence of [SEQ ID NO: 77]; the SCUBE2 second primer has the sequence of [SEQ ID NO: 78]; the SCUBE2 probe oligonucleotide has the sequence of [SEQ ID NO: 79], [SEQ ID NO: 93], or [SEQ ID NO: 95]; the ACTB competitive polynucleotide has the sequence of [SEQ ID NO: 3] or [SEQ ID NO: 2]; the ACTB first primer has the sequence of [SEQ ID NO: 1]; the ACTB second primer has the sequence of [SEQ ID NO: 1] The lymer has the sequence of [SEQ ID NO: 2]; the ACTB probe oligonucleotide has the sequence of [SEQ ID NO: 16] or [SEQ ID NO: 95]; the GAPDH competitive polynucleotide has the sequence of [SEQ ID NO: 6] or [SEQ ID NO: 5]; the GAPDH first primer has the sequence of [SEQ ID NO: 4]; the GAPDH second primer has the sequence of [SEQ ID NO: 5]; the GAPDH probe oligonucleotide has the sequence of [SEQ ID NO: 16] or [SEQ ID NO: 95]; the GUSB competitive polynucleotide has the sequence of [SEQ ID NO: 9] or [SEQ ID NO: 8];The first GUSB primer has the sequence of [SEQ ID NO: 7]; the second GUSB primer has the sequence of [SEQ ID NO: 8]; the GUBS probe oligonucleotide has the sequence of [SEQ ID NO: 16] or [SEQ ID NO: 95]; the RPLP0 competitive polynucleotide has the sequence of [SEQ ID NO: 12] or [SEQ ID NO: 11]; the first RPLP0 primer has the sequence of [SEQ ID NO: 10]; the second RPLP0 primer has the sequence of [SEQ ID NO: 11]; the RPLP0 probe oligonucleotide has the sequence of [SEQ ID NO: 16] or [SEQ ID NO: 95]; the TFRC competitive polynucleotide has the sequence of [SEQ ID NO: 15] or [SEQ ID NO: 14]; the first TFRC primer has the sequence of [SEQ ID NO: 13]; the second TFRC primer has the sequence of [SEQ ID NO: 14]; and / or the TFRC probe oligonucleotide has the sequence of [SEQ ID NO: 16] or [SEQ ID NO: 95].

[0070] In some embodiments, a) Amplification is for the amplification of a housekeeping module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: ACTB, GADPH, GUSB, RPLP0, and TFRC, and the set of competitively amplified polynucleotides is: ii) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and the Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with a Housekeeping Fluorescent Label; or ii) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 3; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 6; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 9; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 12; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 15; and a universal housekeeping module probe oligonucleotide of SEQ ID NO: 16, labeled with a housekeeping fluorescent label, or comprising the same. b) Amplification is for the amplification of an infiltration module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: CD68, CTSV, and MMP11, and the set of competitively amplified polynucleotides is: i) First CD68 primer of SEQ ID NO: 17; second CD68 primer of SEQ ID NO: 18; CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; or ii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 20; and the CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for the infiltration module; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 24; and the CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for the infiltration module; and The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 28; and the MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for the infiltration module. or iii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 81; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 82; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 83; and The present invention comprises or consists of a universal infiltration module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for infiltration modules. c) Amplification is for amplification of a growth module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: AURKA, BIRC5, CCNB1, MK167, and MYBL2, and the set of competitively amplified polynucleotides is: i) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; or ii) The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and the AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for the proliferation module. The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and the BIRC5 probe oligonucleotide of SEQ ID NO: 35, fluorescently labeled for the growth module; The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and the CCNB1 probe oligonucleotide of SEQ ID NO: 39, fluorescently labeled for the proliferation module. The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and the MK167 probe oligonucleotide of SEQ ID NO: 43, fluorescently labeled for the proliferation module. The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; the MYBL2 competitive polynucleotide of SEQ ID NO: 48; and the MYBL2 probe oligonucleotide of SEQ ID NO: 47, fluorescently labeled for the proliferation module. or iii) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 48; and The present invention comprises or consists of a universal proliferation module probe oligonucleotide of SEQ ID NO: 85, labeled with a fluorescent label for proliferation modules. d) Amplification is for the amplification of the HER2 module, which contains or consists of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: ERBB2 and GRB7, and the set of competitively amplified polynucleotides is: i) the first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; ii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 52; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and A universal HER2 module probe oligonucleotide of sequence number 86, labeled with a fluorescent label for HER2 modules; iii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; ERBB2 competitive polynucleotide of SEQ ID NO: 52; and ERBB2 probe oligonucleotide of SEQ ID NO: 51, fluorescently labeled for the HER2 module; The solution comprises or consists of: the first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and the GRB7 probe oligonucleotide of SEQ ID NO: 55, which is labeled with a fluorescent label for the HER2 module. or e) Amplification is for the amplification of an estrogen module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: BAG1, BCL2, ESR1, GSTM1, PGR, and SCUBE2, and the set of competitively amplified polynucleotides is: i) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and A universal estrogen module probe oligonucleotide of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 87; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 88; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 89; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 90; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 91; SEQ ID NO: 77 (first SCUBE2 primer); SEQ ID NO: 78 (second SCUBE2); SEQ ID NO: 92 (SCUBE2 competitive polynucleotide); and A universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 60; and the BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for the estrogen module; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 64; and the BCL2 probe oligonucleotide of SEQ ID NO: 63, fluorescently labeled for the estrogen module. The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 68; and the ESR1 probe oligonucleotide of SEQ ID NO: 67, fluorescently labeled for the estrogen module. The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 72; and the GSTM1 probe oligonucleotide of SEQ ID NO: 71, fluorescently labeled for the estrogen module. The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 76; and the PGR 1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for the estrogen module. The solution comprises or consists of: the first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and the SCUBE2 probe oligonucleotide of SEQ ID NO: 79, labeled with a fluorescent label for the estrogen module.

[0071] In some embodiments, the concentrations of each oligonucleotide are optionally as follows: First ACTB primer of SEQ ID NO: 1 at a concentration of 33 nM or 36 nM; second ACTB primer of SEQ ID NO: 2 at a concentration of 33 nM or 36 nM; ACTB competitive polynucleotide of SEQ ID NO: 3 at a concentration of 1.8 e+02 copies / μL; ACTB competitor of SEQ ID NO: 101 at a concentration of 3.3 e+00 copies / μL; First GADPH primer of SEQ ID NO: 4 at a concentration of 14 nM or 10⁶ nM; second GADPH primer of SEQ ID NO: 5 at a concentration of 14 nM or 10⁶ nM; GADPH competitive polynucleotide of SEQ ID NO: 6 at a concentration of 1 e+0² copies / μL; GADPH competitive polynucleotide of SEQ ID NO: 103 at a concentration of 2.5 e+0² copies / μL; First GUSB primer of SEQ ID NO: 7 at a concentration of 22 nM or 34 nM; second GUSB primer of SEQ ID NO: 8 at a concentration of 22 nM or 24 nM; GUSB competitive polynucleotide of SEQ ID NO: 9 at a concentration of 1.3 e+02 copies / μL; GUSB competitive polynucleotide of SEQ ID NO: 104 at a concentration of 5.2 e+00 copies / μL; First RPLP0 primer of SEQ ID NO: 10 at a concentration of 66 nM or 36 nM; second RPLP0 primer of SEQ ID NO: 11 at a concentration of 66 nM or 36 nM; RPLP0 competitive polynucleotide of SEQ ID NO: 12 at a concentration of 2.9 copies / μL; RPLP0 competitive polynucleotide of SEQ ID NO: 105 at a concentration of 1.5e+01 copies / μL; First TRFC primer of SEQ ID NO: 13 at a concentration of 84 nM or 50 nM; second TRFC primer of SEQ ID NO: 14 at a concentration of 84 nM or 50 nM; TRFC competitive polynucleotide of SEQ ID NO: 15 at a concentration of 1.4 copies / μL; TRFC competitive polynucleotide of SEQ ID NO: 106 at a concentration of 1.4e+01 copies / μL; Universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label, at a concentration of 262 nM; Universal housekeeping module probe oligonucleotide of Sequence ID No. 102, labeled with a housekeeping fluorescent label, at a concentration of 314 nM; A first CD68 primer of SEQ ID NO: 17 at a concentration of 23 nM or 61 nM; a second CD68 primer of SEQ ID NO: 18 at a concentration of 23 nM or 61 nM; a CD68 competitive polynucleotide of SEQ ID NO: 20 at a concentration of 0.6 copies / μL or SEQ ID NO: 110 at a concentration of 1.6e+01 copies / μL; CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for infiltration modules, at a concentration of 28 nM; A first CTSV primer of SEQ ID NO: 21 at a concentration of 28 nM or 52 nM; a second CTSV primer of SEQ ID NO: 22 at a concentration of 28 nM or 52 nM; a CTSV competing polynucleotide of SEQ ID NO: 24 at a concentration of 0.013 copies / μL or of SEQ ID NO: 107 at a concentration of 8.6e-01 copies / μL; CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for infiltration modules, at a concentration of 34 nM; A first MMP11 primer of SEQ ID NO: 25 at a concentration of 20 nM or 82 nM; a second MMP11 primer of SEQ ID NO: 26 at a concentration of 20 nM or 82 nM; an MMP11 competitive polynucleotide of SEQ ID NO: 28 at a concentration of 2.6 copies / μL, or an MMP11 competitive polynucleotide of SEQ ID NO: 109 at a concentration of 1.3e+00 copies / μL; MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for infiltration modules, at a concentration of 24 nM; CD68 competitive polynucleotide of sequence number 81, concentration, 0.6 μg / μL; CTSV competitive polynucleotide of sequence number 82, at a concentration of 0.013 copies / μL; MMP11 competitive polynucleotide of sequence number 83, at a concentration of 2.6 copies / μL; A universal infiltration module probe oligonucleotide of SEQ ID NO: 84, labeled with a fluorescent label for infiltration modules, at a concentration of 86 nM; Universal invasion module probe oligonucleotide of SEQ ID NO: 108, labeled with a fluorescent label for invasion modules, at a concentration of 234 nM; Aurka primer No. 29, first, at a concentration of 17 nM or 26 nM; Aurka primer No. 30, second, at a concentration of 17 nM or 26 nM; Aurka competitive polynucleotide No. 32, at a concentration of 1.5 copies / μL; Aurka competitive polynucleotide No. 111, at a concentration of 6.3e-01 copies / μL; AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for growth modules, at a concentration of 20 nM; First BIRC5 primer of SEQ ID NO: 33 at a concentration of 10 nM or 28 nM; second BIRC5 primer of SEQ ID NO: 34 at a concentration of 10 nM or 28 nM; BIRC5 competitive polynucleotide of SEQ ID NO: 36 at a concentration of 0.13 copies / μL; BIRC5 competitive polynucleotide of SEQ ID NO: 113 at a concentration of 2.9e+00 copies / μL; BIRC5 probe oligonucleotide of SEQ ID NO: 35 at a concentration of 12 nM, labeled with a fluorescent label for the growth module; A first CCNB1 primer of SEQ ID NO: 37 at a concentration of 10 nM or 27 nM; a second CCNB1 primer of SEQ ID NO: 38 at a concentration of 10 nM or 27 nM; a CCNB1 competitive polynucleotide of SEQ ID NO: 40 at a concentration of 0.19 copies / μL; a CCNB1 competitive polynucleotide of SEQ ID NO: 116 at a concentration of 1.0e+01 copies / μL; and a CCNB1 probe oligonucleotide of SEQ ID NO: 39 at a concentration of 12 nM, labeled with a fluorescent label for the growth module. First MK167 primer of SEQ ID NO: 41 at a concentration of 15 nM or 44 nM; second MK167 of SEQ ID NO: 42 at a concentration of 15 nM or 44 nM; MK167 competitive polynucleotide of SEQ ID NO: 44 at a concentration of 0.021 copies / μL; MK167 competitive polynucleotide of SEQ ID NO: 114 at a concentration of 7.4e-01 copies / μL; MK167 probe oligonucleotide of SEQ ID NO: 43 at a concentration of 19 nM, labeled with a fluorescent label for the growth module; First MYBL2 primer of SEQ ID NO: 45 at a concentration of 8 nM or 32 nM; second MYBL2 of SEQ ID NO: 46 at a concentration of 8 nM or 32 nM; MYBL2 competitive polynucleotide of SEQ ID NO: 48 at a concentration of 2.6 e+03 copies / μL; MYBL2 competitive polynucleotide of SEQ ID NO: 115 at a concentration of 4.4 e+00 copies / μL; MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for growth modules, at a concentration of 10 nM; A 73 nM universal proliferation module probe oligonucleotide of SEQ ID NO: 85, labeled with a fluorescent label for proliferation modules; A 188 nM universal proliferation module probe oligonucleotide of SEQ ID NO: 112, labeled with a fluorescent label for proliferation modules; First ERBB2 primer of SEQ ID NO: 49, at a concentration of 15 nM or 20 nM; second ERBB2 of SEQ ID NO: 50, at a concentration of 15 nM or 20 nM; ERBB2 competitive polynucleotide of SEQ ID NO: 52, at a concentration of 0.69 copies / μL, or ERBB2 competitive polynucleotide of SEQ ID NO: 117, at a concentration of 5.3e+00 copies / μL; ERBB2 probe oligonucleotide of SEQ ID NO: 51, at a concentration of 18 nM, labeled with a fluorescent label for the HER2 module; First GRB7 primer of SEQ ID NO: 53 at a concentration of 54 nM or 175 nM; second GRB7 of SEQ ID NO: 54 at a concentration of 54 nM or 175 nM; GRB7 competitive polynucleotide of SEQ ID NO: 56 at a concentration of 40 copies / μL, or GRB7 competitive polynucleotide of SEQ ID NO: 119 at a concentration of 3.6 e-02 copies / μL; GRB7 probe oligonucleotide of SEQ ID NO: 55 at a concentration of 65 nM, labeled with a fluorescent label for the HER2 module; A universal HER2 module probe oligonucleotide of SEQ ID NO: 86, labeled with a fluorescent label for HER2 modules, at a concentration of 83 nM; A 234 nM, HER2 module probe oligonucleotide of Sequence ID No. 118, labeled with a fluorescent label for HER2 modules; A first BAG1 primer of SEQ ID NO: 57 at a concentration of 13 nM or 26 nM; a second BAG1 of SEQ ID NO: 58 at a concentration of 13 nM or 26 nM; a BAG1 competitive polynucleotide of SEQ ID NO: 60 at a concentration of 0.039 copies / μL, or a BAG1 competitive polynucleotide of SEQ ID NO: 100 at a concentration of 1.7e+01 copies / μL; a BAG1 probe oligonucleotide of SEQ ID NO: 59 at a concentration of 16 nM, labeled with a fluorescent label for estrogen modules; A first BCL2 primer of SEQ ID NO: 61 at a concentration of 33 nM or 34 nM; a second BCL2 of SEQ ID NO: 62 at a concentration of 33 nM or 34 nM; a BCL2 competitive polynucleotide of SEQ ID NO: 64 at a concentration of 1.9 copies / μL, or a BCL2 competitive polynucleotide of SEQ ID NO: 97 at a concentration of 1.8e+00 copies / μL; a BCL2 probe oligonucleotide of SEQ ID NO: 63 at a concentration of 39 nM, labeled with a fluorescent label for the estrogen module; First ESR1 primer of SEQ ID NO: 65 at a concentration of 40 nM or 43 nM; second ESR1 of SEQ ID NO: 66 at a concentration of 40 nM or 43 nM; ESR1 competitive polynucleotide of SEQ ID NO: 68 at a concentration of 0.83 copies / μL, or ESR1 competitive polynucleotide of SEQ ID NO: 94 at a concentration of 4.1e+01 copies / μL; ESR1 probe oligonucleotide of SEQ ID NO: 67 at a concentration of 48 nM, labeled with a fluorescent label for estrogen modules; First GSTM1 primer of SEQ ID NO: 69, at a concentration of 11 nM or 52 nM; second GSTM1 of SEQ ID NO: 70, at a concentration of 11 nM or 52 nM; GSTM1 competitive polynucleotide of SEQ ID NO: 72, at a concentration of 0.075 copies / μL, or GSTM1 competitive polynucleotide of SEQ ID NO: 99, at a concentration of 1.9e+00 copies / μL; GSTM1 probe oligonucleotide of SEQ ID NO: 71, at a concentration of 13 nM, labeled with a fluorescent label for estrogen modules; A first PGR primer of SEQ ID NO: 73 at a concentration of 41 nM or 57 nM; a second PGR of SEQ ID NO: 74 at a concentration of 41 nM or 57 nM; a PGR-competitive polynucleotide of SEQ ID NO: 76 at a concentration of 0.015 copies / μL, or a PGR-competitive polynucleotide of SEQ ID NO: 96 at a concentration of 4.4e+00 copies / μL; PGR1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for estrogen modules, at a concentration of 50 nM; First SCUBE2 primer of SEQ ID NO: 77, at a concentration of 22 nM or 54 nM; second SCUBE2 of SEQ ID NO: 78, at a concentration of 22 nM or 54 nM; SCUBE2 competitive polynucleotide of SEQ ID NO: 80, at a concentration of 94 copies / μL, or SCUBE2 competitive polynucleotide of SEQ ID NO: 98, at a concentration of 1.1e+01 copies / μL; SCUBE2 probe oligonucleotide of SEQ ID NO: 79, at a concentration of 26 nM, labeled with a fluorescent label for the estrogen module; BAG1 competitive polynucleotide of sequence number 87, concentration 0.039 copies / μL; BCL2 competitive polynucleotide of sequence number 88, at a concentration of 1.9 copies / μL; ESR1 competitive polynucleotide of SEQ ID NO: 89, concentration 0.83 copies / μL; GSTM1 competitive polynucleotide of SEQ ID NO: 90, concentration 0.075 copies / μL; PGR competitive polynucleotide of SEQ ID NO: 91, concentration 0.015 copies / μL; SCUBE2 competitive polynucleotide of SEQ ID NO: 92 at a concentration of 94 copies / μL; and / or A 192 nM universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; A 319 nM universal estrogen module probe oligonucleotide, labeled with a fluorescent label for estrogen modules, with the code SEQ ID NO: 95.

[0072] In some embodiments, the method is Amplification of at least two of the following modules: housekeeping module, proliferation module, invasion module, HER2 module, and estrogen module; Amplification of at least three of the following modules in the same reaction: housekeeping module, proliferation module, invasion module, HER2 module, and estrogen module; Amplification of at least four of the following modules in the same reaction: housekeeping module, proliferation module, invasion module, HER2 module, and estrogen module; This includes amplification of all five modules in the same reaction: the housekeeping module, the proliferation module, the invasion module, the HER2 module, and the estrogen module. The probe oligonucleotides associated with each module are labeled with the same fluorescent label, and each fluorescent label in the reaction is compatible with other fluorescent labels in the reaction.

[0073] In some embodiments, each probe in the amplification reaction is labeled with the same fluorescent label.

[0074] In some embodiments, each of the five modules is amplified in a separate reaction, and the endpoint fluorescence of each amplified reaction is used for analysis, for example, in the formulas described herein.

[0075] In some embodiments, at least two probes present during the amplification reaction are labeled with different fluorescent labels. In some embodiments, at least three, at least four, or at least five probes present during amplification are each labeled with different fluorophores. In some embodiments, the amplification reaction includes a total of five or fewer different fluorescent labels, optionally four, three, or two or fewer different fluorescent labels. Suitable fluorescent labels are known to those skilled in the art. In some embodiments, the fluorescent labels may be selected from the group including ABY, FAM, JUN, and VIC.

[0076] In some embodiments, multiple target polynucleotides represent the following cDNA polynucleotides: The first module of cDNA polynucleotide; the second module of cDNA polynucleotide; the third module of cDNA polynucleotide; the fourth module of cDNA polynucleotide; the first module of cDNA polynucleotide; the first and second modules of cDNA polynucleotide; the first and third modules of cDNA polynucleotide; the first and fourth modules of cDNA polynucleotide; the first and fifth modules of cDNA polynucleotide; the second and third modules of cDNA polynucleotide; the second and fourth modules of cDNA polynucleotide; the second and fifth modules of cDNA polynucleotide; the third and fourth modules of cDNA polynucleotide; the third and fifth modules of cDNA polynucleotide; the fourth and fifth modules of cDNA polynucleotide; the first, second, and third modules of cDNA polynucleotide; the first, second, and fourth modules of cDNA polynucleotide; The first, second, and fifth modules of creotide; the first, third, and fourth modules of cDNA polynucleotide; the first, third, and fifth modules of cDNA polynucleotide; the first, fourth, and fifth modules of cDNA polynucleotide; the second, third, and fourth modules of cDNA polynucleotide; the second, third, and fifth modules of cDNA polynucleotide; the second, fourth, and fifth modules of cDNA polynucleotide; the third, fourth, and fifth modules of cDNA polynucleotide; the first, second, third, and fourth modules of cDNA polynucleotide; the first, second, third, and fifth modules of cDNA polynucleotide; the first, second, fourth, and fifth modules of cDNA polynucleotide; the first, third, fourth, and fifth modules of cDNA polynucleotide; or the first, second, third, fourth, and fifth modules of cDNA polynucleotide. Here, The first module is a housekeeping module of cDNA polynucleotides derived from mRNA molecules expressed from a group of genes including or consisting of ACTB, GADPH, GUSB, RPLP0, and TFRC; The second module is a cDNA polynucleotide infiltration module derived from mRNA molecules expressed from a group of genes including or consisting of CD68, CTSV, and MMP11; The third module is a cDNA polynucleotide augmentation module derived from mRNA molecules expressed from a group of genes including or consisting of AURKA, CCNB1, MKI67, and MYBL2; The fourth module is a HER2 module of cDNA polynucleotides derived from mRNA molecules expressed from a group of genes including or consisting of ERBB2 and GRB7; and The fifth module is an estrogen module of cDNA polynucleotides derived from mRNA molecules expressed from a group of genes including or consisting of BAG1, BCL2, ESR1, GSTM1, PGY, and SCUBE2.

[0077] In some embodiments, Each probe oligonucleotide in the set of target-competitive amplification oligonucleotides corresponding to each target in the first module contains the same fluorescent label for the first module; Each probe oligonucleotide in the set of target-competitive amplification oligonucleotides corresponding to each target in the second module contains the same fluorescent label for the second module; Each probe oligonucleotide in the set of target-competitive amplification oligonucleotides corresponding to each target in the third module contains the same fluorescent label for the third module; Each probe oligonucleotide in the set of target-competitive amplification oligonucleotides corresponding to each target in the fourth module contains the same fluorescent label for the fourth module; and / or Each probe oligonucleotide in the set of target-competitive amplification oligonucleotides corresponding to each target in the fifth module contains the same fluorescent label for the fifth module.

[0078] In some embodiments, the housekeeping fluorescent labels, proliferation fluorescent labels, invasion fluorescent labels, HER2 fluorescent labels, and fluorescent labels for the estrogen module are all different fluorescent labels, optionally interchangeable fluorescent labels.

[0079] In some embodiments, Any two of the following are the same fluorescent label: housekeeping fluorescent label, proliferation fluorescent label, invasion fluorescent label, HER2 fluorescent label, and estrogen module fluorescent label; Any three of the following are the same fluorescent label: housekeeping fluorescent label, proliferation fluorescent label, invasion fluorescent label, HER2 fluorescent label, and estrogen module fluorescent label; Any four of the following are the same fluorescent label: housekeeping fluorescent label, proliferation fluorescent label, invasion fluorescent label, HER2 fluorescent label, and estrogen module fluorescent label; or Housekeeping fluorescent labels, proliferation fluorescent labels, infiltration fluorescent labels, HER2 fluorescent labels, and estrogen module fluorescent labels are all the same fluorescent labels.

[0080] In some embodiments, the method includes a step prior to step (a) in which a target polynucleotide cDNA molecule is prepared by reverse transcription from mRNA polynucleotide, and optionally, the reverse transcription step occurs in a single reverse transcription-qPCR step (rt-qPCR). Methods for preparing cDNA molecules from mRNA by reverse transcription are known to those skilled in the art.

[0081] In some embodiments, the reverse transcription step is performed independently of at least two, i.e., multiple, target polynucleotide amplification methods for generating cDNA used in the amplification method.

[0082] In some embodiments, the sample obtained from the subject is a tumor specimen, optionally a breast tumor biopsy. Appropriate biopsy methods are known to those skilled in the art.

[0083] Any amplification method is suitable for use with the present invention. However, preferred amplification methods include polymerase chain reaction (PCR) or recombinase polymerase reaction (RPA). In some embodiments, amplification is PCR.

[0084] In some embodiments, the amplification reaction is a PCR-based reaction and is carried out for a specific number of cycles, at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, or 60 cycles, at any choice.

[0085] In some embodiments, the method further includes determining the fluorescence signal intensity of each fluorescent label associated with the probe oligonucleotide present in the amplified reactant produced after a specific number of cycles. In some embodiments, the fluorescence signal intensity of each fluorescent label associated with the probe oligonucleotide present in the amplified reactant is determined after 50 amplification cycles.

[0086] The present invention also provides a method for generating a score (risk score or RS) for use in determining the postoperative risk of breast cancer recurrence and / or predicting the response to chemotherapy, the method comprising a method for amplifying at least two, i.e., multiple, target polynucleotides in a sample provided herein. Preferred embodiments of the method for generating a score for use in determining the postoperative risk of breast cancer recurrence and / or predicting the response to chemotherapy are as described elsewhere in the context of the amplification method of the present invention.

[0087] In some embodiments, the method is a) Amplification of a housekeeping module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: ACTB, GADPH, GUSB, RPLP0, and TFRC, wherein the set of competitively amplified polynucleotides used for amplification is: i) First ACTB primer of SEQ ID NO: 1; second ACTB primer of SEQ ID NO: 2; ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with Housekeeping Fluorescent Label. or ii) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 3; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 6; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 9; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 12; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 15; and Amplification comprising or consisting of the universal housekeeping module probe oligonucleotide of SEQ ID NO: 16, labeled with a housekeeping fluorescent label; and b) Amplification of an infiltration module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: CD68, CTSV, and MMP11, wherein the set of competitively amplified polynucleotides used for amplification is: i) First CD68 primer of SEQ ID NO: 17; second CD68 primer of SEQ ID NO: 18; CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; or ii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 20; and the CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for the infiltration module; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 24; and the CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for the infiltration module. The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 28; and the MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for the infiltration module. or iii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 81; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 82; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 83; and A universal infiltration module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for infiltration modules, or comprising the above; and c) Amplification of a growth module containing or comprising target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the following genes: AURKA, BIRC5, CCNB1, MK167, and MYBL2, wherein the set of competitively amplified polynucleotides used for amplification is: i) the first AURA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 primer of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; or ii) The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and the AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for the proliferation module. The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and the BIRC5 probe oligonucleotide of SEQ ID NO: 35, fluorescently labeled for the growth module; The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and the CCNB1 probe oligonucleotide of SEQ ID NO: 39, fluorescently labeled for the proliferation module. The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and the MK167 probe oligonucleotide of SEQ ID NO: 43, fluorescently labeled for the proliferation module. The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; the MYBL2 competitive polynucleotide of SEQ ID NO: 48; and the MYBL2 probe oligonucleotide of SEQ ID NO: 47, fluorescently labeled for the proliferation module. or iii) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 48; and Includes or consists of a universal proliferation module probe oligonucleotide of Sequence ID No. 85, labeled with a fluorescent label for proliferation modules; and d) Amplification of a HER2 module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or consisting of the following genes: ERBB2 and GRB7, wherein the set of competitively amplified polynucleotides used for amplification is: i) the first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; ii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 52; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and A universal HER2 module probe oligonucleotide of sequence number 86, labeled with a fluorescent label for HER2 modules; iii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; ERBB2 competitive polynucleotide of SEQ ID NO: 52; and ERBB2 probe oligonucleotide of SEQ ID NO: 51, fluorescently labeled for the HER2 module; It comprises or consists of: the first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and the GRB7 probe oligonucleotide of SEQ ID NO: 55, which is labeled with a fluorescent label for the HER2 module; and e) Amplification of an estrogen module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: BAG1, BCL2, ESR1, GSTM1, PGR, and SCUBE2, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and A universal estrogen module probe oligonucleotide of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 87; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 88; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 89; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 90; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 91; SEQ ID NO: 77 (first SCUBE2 primer); SEQ ID NO: 78 (second SCUBE2); SEQ ID NO: 92 (SCUBE2 competitive polynucleotide); and A universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 60; and the BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for the estrogen module; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 64; and the BCL2 probe oligonucleotide of SEQ ID NO: 63, fluorescently labeled for the estrogen module. The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 68; and the ESR1 probe oligonucleotide of SEQ ID NO: 67, fluorescently labeled for the estrogen module. The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 72; and the GSTM1 probe oligonucleotide of SEQ ID NO: 71, fluorescently labeled for the estrogen module. The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 76; and the PGR 1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for the estrogen module. The solution comprises or consists of: the first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and the SCUBE2 probe oligonucleotide of SEQ ID NO: 79, labeled with a fluorescent label for the estrogen module.

[0088] The risk score, or RS, is a number between 0 and 100 and is used to predict the risk of breast cancer recurring in distant sites and whether chemotherapy may help reduce that risk. The RS provided by this invention should be interpreted in the same manner as the RS provided by the Oncotype DX trial. See, for example, the Oncotype website https: / / www.oncotypeiq.com, e.g., https: / / www.oncotypeiq.com / en-gb / breast-cancer / healthcare-professionals / oncotype-dx-breast-recurrence-score / interpreting-the-results;Breast J.2022;2022:1199245;Breast Cancer Res Treat.2020;180(3):809-817. Interpretation and guidance for ongoing clinical interventions may change over time (see, for example, Cancers (Basel).2023 Jun;15(12):3217). This trial also provides the percentage of the risk of breast cancer returning to another part of the body as a “distant recurrence” within nine years if treated with hormone therapy alone for five years. The trial also provides the percentage of the expected benefit of adding chemotherapy to hormone therapy to reduce the risk of cancer recurrence or death for the recurrence score group.

[0089] For example, in lymph node-negative patients, an RS score of 0-25 generally indicates no benefit from adding chemotherapy to endocrine therapy. In patients with an RS score of 26-100, the group as a whole is considered to have a significant (26%) benefit from adding chemotherapy to endocrine therapy.

[0090] In patients with lymph node metastasis: Patients with a recurrence score of 0-17 will not benefit from adding chemotherapy to endocrine therapy. Patients with a recurrence score of 18-30 may benefit from adding chemotherapy to endocrine therapy. Patients with a relapse score of 31-100 would significantly benefit from adding chemotherapy to endocrine therapy.

[0091] Furthermore, methods for determining the postoperative risk of breast cancer recurrence and / or predicting the response to chemotherapy are also provided herein, which include methods for generating a score for use in determining the postoperative risk of breast cancer recurrence and / or predicting the response to chemotherapy provided herein, or methods for amplifying at least two, i.e., multiple, target polynucleotides in a sample provided herein.

[0092] In some embodiments, the sample obtained from the subject is a breast tumor biopsy.

[0093] In some embodiments, the subject is HR+HER2-positive breast cancer; Having or having previously had invasive breast cancer of anatomical stage I, II, or IIIa; and / or The test is positive for cancer cells in 0, 1, 2, or 3 or fewer axillary lymph nodes.

[0094] In some embodiments, the concentrations of each oligonucleotide in the amplification reaction are optionally as follows: First ACTB primer of SEQ ID NO: 1 at a concentration of 33 nM or 36 nM; second ACTB primer of SEQ ID NO: 2 at a concentration of 33 nM or 36 nM; ACTB competitive polynucleotide of SEQ ID NO: 3 at a concentration of 1.8 e+02 copies / μL; ACTB competitor of SEQ ID NO: 101 at a concentration of 3.3 e+00 copies / μL; A first GADPH primer of SEQ ID NO: 4 at a concentration of 14 nM or 10⁶ nM; a second GADPH primer of SEQ ID NO: 5 at a concentration of 14 nM or 10⁶ nM; a GADPH-competitive polynucleotide of SEQ ID NO: 6 at a concentration of 1 e+0² copies / μL; a GADPH-competitive polynucleotide of SEQ ID NO: 103 at a concentration of 2.5 e+0² copies / μL; First GUSB primer of SEQ ID NO: 7 at a concentration of 22 nM or 34 nM; second GUSB primer of SEQ ID NO: 8 at a concentration of 22 nM or 24 nM; GUSB competitive polynucleotide of SEQ ID NO: 9 at a concentration of 1.3 e+02 copies / μL; GUSB competitive polynucleotide of SEQ ID NO: 104 at a concentration of 5.2 e+00 copies / μL; First RPLP0 primer of SEQ ID NO: 10 at a concentration of 66 nM or 36 nM; second RPLP0 primer of SEQ ID NO: 11 at a concentration of 66 nM or 36 nM; RPLP0 competitive polynucleotide of SEQ ID NO: 12 at a concentration of 2.9 copies / μL; RPLP0 competitive polynucleotide of SEQ ID NO: 105 at a concentration of 1.5e+01 copies / μL; First TRFC primer of SEQ ID NO: 13 at a concentration of 84 nM or 50 nM; second TRFC primer of SEQ ID NO: 14 at a concentration of 84 nM or 50 nM; TRFC competitive polynucleotide of SEQ ID NO: 15 at a concentration of 1.4 copies / μL; TRFC competitive polynucleotide of SEQ ID NO: 106 at a concentration of 1.4e+01 copies / μL; Universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label, at a concentration of 262 nM; Universal housekeeping module probe oligonucleotide of Sequence ID No. 102, labeled with a housekeeping fluorescent label, at a concentration of 314 nM; A first CD68 primer of SEQ ID NO: 17 at a concentration of 23 nM or 61 nM; a second CD68 primer of SEQ ID NO: 18 at a concentration of 23 nM or 61 nM; a CD68 competitive polynucleotide of SEQ ID NO: 20 at a concentration of 0.6 copies / μL or of SEQ ID NO: 110 at a concentration of 1.6e+01 copies / μL; CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for infiltration modules, at a concentration of 28 nM; A first CTSV primer of SEQ ID NO: 21 at a concentration of 28 nM or 52 nM; a second CTSV primer of SEQ ID NO: 22 at a concentration of 28 nM or 52 nM; a CTSV competitive polynucleotide of SEQ ID NO: 24 at a concentration of 0.013 copies / μL or of SEQ ID NO: 107 at a concentration of 8.6e-01 copies / μL; CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for infiltration modules, at a concentration of 34 nM; A first MMP11 primer of SEQ ID NO: 25 at a concentration of 20 nM or 82 nM; a second MMP11 primer of SEQ ID NO: 26 at a concentration of 20 nM or 82 nM; MMP11 competitive polynucleotide of SEQ ID NO: 28 at a concentration of 2.6 copies / μL, or MMP11 competitive polynucleotide of SEQ ID NO: 109 at a concentration of 1.3e+00 copies / μL; MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for infiltration modules, at a concentration of 24 nM; CD68 competitive polynucleotide of sequence number 81, concentration, 0.6 μg / μL; CTSV competitive polynucleotide of sequence number 82 at a concentration of 0.013 copies / μL; MMP11 competitive polynucleotide of sequence number 83, at a concentration of 2.6 copies / μL; A universal infiltration module probe oligonucleotide of SEQ ID NO: 84, labeled with a fluorescent label for infiltration modules, at a concentration of 86 nM; A 234 nM universal infiltration module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for infiltration modules; Aurka primer No. 29, first, at a concentration of 17 nM or 26 nM; Aurka primer No. 30, second, at a concentration of 17 nM or 26 nM; Aurka competitive polynucleotide No. 32, at a concentration of 1.5 copies / μL; Aurka competitive polynucleotide No. 111, at a concentration of 6.3e-01 copies / μL; AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for growth modules, at a concentration of 20 nM; First BIRC5 primer of SEQ ID NO: 33 at a concentration of 10 nM or 28 nM; second BIRC5 primer of SEQ ID NO: 34 at a concentration of 10 nM or 28 nM; BIRC5 competitive polynucleotide of SEQ ID NO: 36 at a concentration of 0.13 copies / μL; BIRC5 competitive polynucleotide of SEQ ID NO: 113 at a concentration of 2.9e+00 copies / μL; BIRC5 probe oligonucleotide of SEQ ID NO: 35 at a concentration of 12 nM, labeled with a fluorescent label for the growth module; A first CCNB1 primer of SEQ ID NO: 37 at a concentration of 10 nM or 27 nM; a second CCNB1 primer of SEQ ID NO: 38 at a concentration of 10 nM or 27 nM; a CCNB1 competitive polynucleotide of SEQ ID NO: 40 at a concentration of 0.19 copies / μL; a CCNB1 competitive polynucleotide of SEQ ID NO: 116 at a concentration of 1.0e+01 copies / μL; and a CCNB1 probe oligonucleotide of SEQ ID NO: 39 at a concentration of 12 nM, labeled with a fluorescent label for the growth module. First MK167 primer of SEQ ID NO: 41 at a concentration of 15 nM or 44 nM; second MK167 of SEQ ID NO: 42 at a concentration of 15 nM or 44 nM; MK167 competitive polynucleotide of SEQ ID NO: 44 at a concentration of 0.021 copies / μL; MK167 competitive polynucleotide of SEQ ID NO: 114 at a concentration of 7.4e-01 copies / μL; MK167 probe oligonucleotide of SEQ ID NO: 43 at a concentration of 19 nM, labeled with a fluorescent label for the growth module; First MYBL2 primer of SEQ ID NO: 45 at a concentration of 8 nM or 32 nM; second MYBL2 of SEQ ID NO: 46 at a concentration of 8 nM or 32 nM; MYBL2 competitive polynucleotide of SEQ ID NO: 48 at a concentration of 2.6 e+03 copies / μL; MYBL2 competitive polynucleotide of SEQ ID NO: 115 at a concentration of 4.4 e+00 copies / μL; MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for growth modules, at a concentration of 10 nM; A 73 nM universal proliferation module probe oligonucleotide of SEQ ID NO: 85, labeled with a fluorescent label for proliferation modules; A 188 nM universal proliferation module probe oligonucleotide of SEQ ID NO: 112, labeled with a fluorescent label for proliferation modules; First ERBB2 primer of SEQ ID NO: 49 at a concentration of 15 nM or 20 nM; second ERBB2 of SEQ ID NO: 50 at a concentration of 15 nM or 20 nM; ERBB2 competitive polynucleotide of SEQ ID NO: 52 at a concentration of 0.69 copies / μL, or ERBB2 competitive polynucleotide of SEQ ID NO: 117 at a concentration of 5.3e+00 copies / μL; ERBB2 probe oligonucleotide of SEQ ID NO: 51 at a concentration of 18 nM, labeled with a fluorescent label for the HER2 module; First GRB7 primer of SEQ ID NO: 53 at a concentration of 54 nM or 175 nM; second GRB7 of SEQ ID NO: 54 at a concentration of 54 nM or 175 nM; GRB7 competitive polynucleotide of SEQ ID NO: 56 at a concentration of 40 copies / μL, or GRB7 competitive polynucleotide of SEQ ID NO: 119 at a concentration of 3.6 e-02 copies / μL; GRB7 probe oligonucleotide of SEQ ID NO: 55 at a concentration of 65 nM, labeled with a fluorescent label for the HER2 module; A universal HER2 module probe oligonucleotide of SEQ ID NO: 86, labeled with a fluorescent label for HER2 modules, at a concentration of 83 nM; A 234 nM, HER2 module probe oligonucleotide of Sequence ID No. 118, labeled with a fluorescent label for HER2 modules; A first BAG1 primer of SEQ ID NO: 57 at a concentration of 13 nM or 26 nM; a second BAG1 of SEQ ID NO: 58 at a concentration of 13 nM or 26 nM; a BAG1 competitive polynucleotide of SEQ ID NO: 60 at a concentration of 0.039 copies / μL, or a BAG1 competitive polynucleotide of SEQ ID NO: 100 at a concentration of 1.7e+01 copies / μL; a BAG1 probe oligonucleotide of SEQ ID NO: 59 at a concentration of 16 nM, labeled with a fluorescent label for estrogen modules; A first BCL2 primer of SEQ ID NO: 61 at a concentration of 33 nM or 34 nM; a second BCL2 of SEQ ID NO: 62 at a concentration of 33 nM or 34 nM; a BCL2 competitive polynucleotide of SEQ ID NO: 64 at a concentration of 1.9 copies / μL, or a BCL2 competitive polynucleotide of SEQ ID NO: 97 at a concentration of 1.8e+00 copies / μL; a BCL2 probe oligonucleotide of SEQ ID NO: 63 at a concentration of 39 nM, labeled with a fluorescent label for the estrogen module; First ESR1 primer of SEQ ID NO: 65 at a concentration of 40 nM or 43 nM; second ESR1 of SEQ ID NO: 66 at a concentration of 40 nM or 43 nM; ESR1 competitive polynucleotide of SEQ ID NO: 68 at a concentration of 0.83 copies / μL, or ESR1 competitive polynucleotide of SEQ ID NO: 94 at a concentration of 4.1e+01 copies / μL; ESR1 probe oligonucleotide of SEQ ID NO: 67 at a concentration of 48 nM, labeled with a fluorescent label for estrogen modules; First GSTM1 primer of SEQ ID NO: 69 at a concentration of 11 nM or 52 nM; second GSTM1 of SEQ ID NO: 70 at a concentration of 11 nM or 52 nM; GSTM1 competitive polynucleotide of SEQ ID NO: 72 at a concentration of 0.075 copies / μL, or GSTM1 competitive polynucleotide of SEQ ID NO: 99 at a concentration of 1.9e+00 copies / μL; GSTM1 probe oligonucleotide of SEQ ID NO: 71 at a concentration of 13 nM, labeled with a fluorescent label for estrogen modules; A first PGR primer of SEQ ID NO: 73 at a concentration of 41 nM or 57 nM; a second PGR of SEQ ID NO: 74 at a concentration of 41 nM or 57 nM; a PGR-competitive polynucleotide of SEQ ID NO: 76 at a concentration of 0.015 copies / μL, or a PGR-competitive polynucleotide of SEQ ID NO: 96 at a concentration of 4.4e+00 copies / μL; PGR1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for estrogen modules, at a concentration of 50 nM; First SCUBE2 primer of SEQ ID NO: 77, at a concentration of 22 nM or 54 nM; second SCUBE2 of SEQ ID NO: 78, at a concentration of 22 nM or 54 nM; SCUBE2 competitive polynucleotide of SEQ ID NO: 80, at a concentration of 94 copies / μL, or SCUBE2 competitive polynucleotide of SEQ ID NO: 98, at a concentration of 1.1e+01 copies / μL; SCUBE2 probe oligonucleotide of SEQ ID NO: 79, at a concentration of 26 nM, labeled with a fluorescent label for the estrogen module; BAG1 competitive polynucleotide of sequence number 87, concentration 0.039 copies / μL; BCL2 competitive polynucleotide of sequence number 88, at a concentration of 1.9 copies / μL; ESR1 competitive polynucleotide of SEQ ID NO: 89, concentration 0.83 copies / μL; GSTM1 competitive polynucleotide of SEQ ID NO: 90, concentration 0.075 copies / μL; PGR competitive polynucleotide of SEQ ID NO: 91 at a concentration of 0.015 copies / μL; SCUBE2 competitive polynucleotide of SEQ ID NO: 92 at a concentration of 94 copies / μL; and / or A 192 nM universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; A 319 nM universal estrogen module probe oligonucleotide, labeled with a fluorescent label for estrogen modules, with the code SEQ ID NO: 95.

[0095] In some embodiments, the method includes carrying out five separate amplification reactions: a) A first amplification reaction for the infiltration module, wherein a set of competing oligonucleotides: i) First CD68 primer of SEQ ID NO: 17; second CD68 primer of SEQ ID NO: 18; CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and comprising or consisting of a universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; or ii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 20; and the CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for the infiltration module; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 24; and the CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for the infiltration module. The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 28; and the MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for the infiltration module. or iii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 81; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 82; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 83; and A universal infiltration module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for infiltration modules, or comprising the above; or iii) A CD68 competitive polynucleotide having the sequence of [SEQ ID NO: 81] (optional); a first CD68 primer having the sequence of [SEQ ID NO: 17] (optional); a second CD68 primer having the sequence of [SEQ ID NO: 18] (optional); Optionally, a CTSV-competitive polynucleotide having the sequence of [SEQ ID NO: 82]; optionally, a first CTSV primer having the sequence of [SEQ ID NO: 21]; optionally, a second CTSV primer having the sequence of [SEQ ID NO: 22]; A combination of an optional MP11 competitive polynucleotide having the sequence of [SEQ ID NO: 83]; an optional first MP11 primer having the sequence of [SEQ ID NO: 25]; an optional second MP11 primer having the sequence of [SEQ ID NO: 26]; and a universal infiltration module probe oligonucleotide of SEQ ID NO: 84 labeled with a fluorescent label for infiltration modules; b) A second amplification reaction for the proliferation module, wherein a set of competing oligonucleotides: i) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Includes or consists of a universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; or ii) The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and the AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for the proliferation module. The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and the BIRC5 probe oligonucleotide of SEQ ID NO: 35, fluorescently labeled for the growth module; The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and the CCNB1 probe oligonucleotide of SEQ ID NO: 39, fluorescently labeled for the proliferation module. The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and the MK167 probe oligonucleotide of SEQ ID NO: 43, fluorescently labeled for the proliferation module. The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; the MYBL2 competitive polynucleotide of SEQ ID NO: 48; and the MYBL2 probe oligonucleotide of SEQ ID NO: 47, fluorescently labeled for the proliferation module. or iii) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 48; and Includes or comprises a universal proliferation module probe oligonucleotide of SEQ ID NO: 85, labeled with a fluorescent label for proliferation modules, All probe oligonucleotides are labeled with fluorescent labels for the growth module. c) A third amplification reaction for the Her2 module of the target polynucleotide, wherein a set of competing oligonucleotides: i) the first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; or ii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 52; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and comprising or consisting of a universal HER2 module probe oligonucleotide of sequence number 86, labeled with a fluorescent label for HER2 modules; or iii) The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; ERBB2 competitive polynucleotide of SEQ ID NO: 52; and ERBB2 probe oligonucleotide of SEQ ID NO: 51, fluorescently labeled for the HER2 module; It comprises or consists of: the first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and the GRB7 probe oligonucleotide of SEQ ID NO: 55, which is labeled with a fluorescent label for the HER2 module; d) A fourth amplification reaction for the estrogen module of the target polynucleotide, wherein a set of competing oligonucleotides: i) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and Includes or comprises a universal estrogen module probe oligonucleotide of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 87; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 88; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 89; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 90; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 91; SEQ ID NO: 77 (first SCUBE2 primer); SEQ ID NO: 78 (second SCUBE2); SEQ ID NO: 92 (SCUBE2 competitive polynucleotide); and Includes or comprises a universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 60; and the BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for the estrogen module; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 64; and the BCL2 probe oligonucleotide of SEQ ID NO: 63, fluorescently labeled for the estrogen module. The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 68; and the ESR1 probe oligonucleotide of SEQ ID NO: 67, fluorescently labeled for the estrogen module. The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 72; and the GSTM1 probe oligonucleotide of SEQ ID NO: 71, fluorescently labeled for the estrogen module. The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 76; and the PGR 1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for the estrogen module. The first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and the SCUBE2 probe oligonucleotide of SEQ ID NO: 79, labeled with a fluorescent label for the estrogen module, comprising or comprising these; and e) A fifth amplification reaction for the housekeeping module of the target polynucleotide, wherein a set of competing oligonucleotides: i) First ACTB primer of SEQ ID NO: 1; second ACTB primer of SEQ ID NO: 2; ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and a universal housekeeping module probe oligonucleotide of sequence number 102 labeled with a housekeeping fluorescent label, or comprising the same; or ii) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 3; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 6; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 9; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 12; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 15; and The universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label, is provided with or comprises such oligonucleotides.

[0096] In a preferred embodiment, the housekeeping module is amplified using the following oligonucleotides: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and a universal housekeeping module probe oligonucleotide of sequence number 102, labeled with a housekeeping fluorescent label.

[0097] In a preferred embodiment, the infiltration module is amplified using the following oligonucleotides: The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and A universal infiltration module probe oligonucleotide of sequence number 108, labeled with a fluorescent label for infiltration modules.

[0098] In a preferred embodiment, the proliferation module is amplified using the following oligonucleotides: The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and A universal proliferation module probe oligonucleotide of sequence number 112, labeled with a fluorescent label for proliferation modules.

[0099] In a preferred embodiment, the Her2 module is amplified using the following oligonucleotides: The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules.

[0100] In a preferred embodiment, the estrogen module is amplified using the following oligonucleotides: The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and A universal estrogen module probe oligonucleotide of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules.

[0101] In a more preferred method, the method comprises five distinct modular reactions, which are: Amplification of the housekeeping module using the following oligonucleotides: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and the Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with a Housekeeping Fluorescent Label; and The infiltration module is amplified using the following oligonucleotides: The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; and The amplification of the proliferation module is performed using the following oligonucleotides: The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; and The Her2 module is amplified using the following oligonucleotides: The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; and The estrogen module is amplified using the following oligonucleotides: The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and Universal estrogen module probe oligonucleotide.

[0102] Those skilled in the art will understand that any of the methods described herein may include a further step of determining an appropriate treatment profile for the subject on which the method was performed (on a sample obtained from the subject). In some cases where the risk score indicates a low probability of cancer recurrence, the treatment protocol may simply involve monitoring the patient, for example, through periodic scans or repetitions of the methods described herein. In other cases, the risk profile may indicate that further therapeutic agents or surgery, including chemotherapy and radiotherapy, may be justified.

[0103] The present invention also provides a method for treating breast cancer in a subject or preventing the recurrence of breast cancer, wherein the risk profile for breast cancer recurrence is determined for a sample obtained from the subject according to one of the methods of the present invention.

[0104] The present invention also provides a kit for carrying out this preferred embodiment.

[0105] In some embodiments, the invasion module label or fluorophore, the proliferation module label or fluorophore, the housekeeping module fluorophore, the estrogen module label or fluorophore, and the Her2 module label or fluorophore are all different, for example, all having different spectral characteristics. In other embodiments, the invasion module label or fluorophore, the proliferation module label or fluorophore, the housekeeping module fluorophore, the estrogen module label or fluorophore, and the Her2 module label or fluorophore are all the same label or fluorophore.

[0106] In some embodiments, the amplification reaction is a PCR reaction.

[0107] In some embodiments, each distinct amplification reaction is performed for 50 cycles, and the fluorescence signal intensity of each of the invasion module label or fluorophore, proliferation module label or fluorophore, housekeeping module fluorophore, estrogen module label or fluorophore, and Her2 module label or fluorophore is determined.

[0108] In some embodiments, the PCR reaction is performed for 50 cycles.

[0109] In some embodiments, the fluorescence signal intensity of each fluorophore is determined after a set number of amplification cycles, optionally after 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, or 60 cycles.

[0110] In some embodiments, the fluorescence signal intensity of each fluorophore is determined after 50 amplification cycles.

[0111] In one aspect, provided herein is a kit comprising at least two polynucleotides provided herein. In one embodiment, the kit comprises at least two polynucleotides each having any one of the sequences of SEQ ID NO: 1 to 98.

[0112] The present invention also provides a kit comprising any one, two or more, or all of the following groups of polynucleotides: a) i) the first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 3; the first GAPDH primer of SEQ ID NO: 4; the second GAPDH primer of SEQ ID NO: 5; the GAPDH competitive polynucleotide of SEQ ID NO: 6; the first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 9; the first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 12; the first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 15; and the universal housekeeping module probe oligonucleotide of SEQ ID NO: 16 labeled with a housekeeping fluorescent label, comprising or consisting thereof; or ii) the first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; the first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; the first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; the first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 105; the first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and the universal housekeeping module probe oligonucleotide of SEQ ID NO: 102 labeled with a housekeeping fluorescent label; b) i) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of SEQ ID NO: 20; and CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for infiltration modules; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competitive polynucleotide of sequence number 24; and CTSV probe oligonucleotide of sequence number 23, labeled with a fluorescent label for infiltration modules; and The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of SEQ ID NO: 28; and MMP11 probe oligonucleotide of sequence number 27, labeled with a fluorescent label for infiltration modules; or ii) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 81; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competitive polynucleotide of sequence number 82; The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of sequence number 83; and Universal invasion module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for invasion modules; or iii) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; The CD68 competitive polynucleotide of SEQ ID NO: 108; The first CTSV primer of SEQ ID NO: 21; The second CTSV primer of SEQ ID NO: 22; The CTSV competitive polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; The second MMP11 primer of SEQ ID NO: 26; The MMP11 competitive polynucleotide of SEQ ID NO: 109; and The universal invasion module probe oligonucleotide of SEQ ID NO: 108 labeled with a fluorescent label for the invasion module; c) Amplification of a proliferation module comprising or consisting of a target polynucleotide which is a cDNA molecule derived from an mRNA polynucleotide expressed from the group comprising or consisting of the following genes: AURKA, BIRC5, CCNB1, MK167 and MYBL2, wherein the set of competitive amplification polynucleotides used for the amplification is: i) The first AURKA primer of SEQ ID NO: 29; The second AURKA primer of SEQ ID NO: 30; The AURKA competitive polynucleotide of SEQ ID NO: 32; and The AURKA probe oligonucleotide of SEQ ID NO: 31 labeled with a fluorescent label for the proliferation module; The first BIRC5 primer of SEQ ID NO: 33; The second BIRC5 primer of SEQ ID NO: 34; The BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The BIRC5 probe oligonucleotide of SEQ ID NO: 35 labeled with a fluorescent label for the proliferation module; The first CCNB1 primer of SEQ ID NO: 37; The second CCNB1 primer of SEQ ID NO: 38; The CCNB1 competitive polynucleotide of SEQ ID NO: 40; and The CCNB1 probe oligonucleotide of SEQ ID NO: 39 labeled with a fluorescent label for the proliferation module; The first MK167 primer of sequence number 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of SEQ ID NO: 44; and MK167 probe oligonucleotide of SEQ ID NO: 43, labeled with a fluorescent label for a growth module; The first MYBL2 primer of Sequence ID No. 45; The second MYBL2 in sequence number 46; MYBL2 competitive polynucleotide of sequence number 48; and Containing or comprising the MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for the proliferation module; or ii) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 36; and CCNB1 primer No. 1 of Sequence ID No. 37; The second CCNB1 primer of sequence number 38; CCNB1 competitive polynucleotide of sequence number 40; and The first MK167 primer of sequence number 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of SEQ ID NO: 44; and The first MYBL2 primer of Sequence ID No. 45; The second MYBL2 in sequence number 46; MYBL2 competitive polynucleotide of sequence number 48; and Includes or consists of a universal proliferation module probe oligonucleotide of Sequence ID No. 85, labeled with a fluorescent label for proliferation modules; or iii) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of sequence number 111; and The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of sequence number 113; and CCNB1 primer No. 1 of Sequence ID No. 37; The second CCNB1 primer of sequence number 38; CCNB1 competitive polynucleotide of sequence number 116; and The first MK167 primer of sequence number 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of sequence number 114; and The first MYBL2 primer of Sequence ID No. 45; The second MYBL2 in sequence number 46; MYBL2 competitive polynucleotide of sequence number 115; and Includes or consists of a universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; d) Amplification of a HER2 module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or consisting of the following genes: ERBB2 and GRB7, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of Sequence ID No. 52; and ERBB2 probe oligonucleotide of sequence number 51, labeled with a fluorescent label for HER2 modules; The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; The GRB7 competitive polynucleotide of SEQ ID NO: 56; and Containing or comprising the GRB7 probe oligonucleotide of sequence number 55, labeled with a fluorescent label for the HER2 module; or ii) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of Sequence ID No. 52; and The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; The GRB7 competitive polynucleotide of SEQ ID NO: 56; and comprising or consisting of a universal HER2 module probe oligonucleotide of sequence number 86, labeled with a fluorescent label for HER2 modules; or iii) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of sequence number 117; and The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of sequence number 119; and Includes or consists of a universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; and / or e) Amplification of an estrogen module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: BAG1, BCL2, ESR1, GSTM1, PGR, and SCUBE2, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first BAG1 primer of Sequence ID No. 57; The second BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 60; and BAG1 probe oligonucleotide of SEQ ID NO: 59, fluorescently labeled for estrogen modules; The first BCL2 primer of Sequence ID No. 61; The second BCL2 of sequence number 62; BCL2 competitive polynucleotide of SEQ ID NO: 64; and BCL2 probe oligonucleotide of SEQ ID NO: 63, labeled with a fluorescent label for estrogen modules; The first ESR1 primer of Sequence ID No. 65; The second ESR1 in sequence number 66; ESR1 competitive polynucleotide of SEQ ID NO: 68; and ESR1 probe oligonucleotide of SEQ ID NO: 67, labeled with a fluorescent label for estrogen modules; The first GSTM1 primer of sequence number 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 72; and GSTM1 probe oligonucleotide of SEQ ID NO: 71, fluorescently labeled for estrogen modules; PGR primer No. 1 of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of SEQ ID NO: 76; and PGR1 probe oligonucleotide of SEQ ID NO: 75, fluorescently labeled for estrogen modules; SCUBE2 primer No. 1 of Sequence ID No. 77; SCUBE2, the second element of sequence number 78; SCUBE2 competitive polynucleotide of sequence number 80; and Containing or comprising SCUBE2 probe oligonucleotides of SEQ ID NO: 79, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of Sequence ID No. 57; The second BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 87; The first BCL2 primer of Sequence ID No. 61; The second BCL2 of sequence number 62; BCL2 competitive polynucleotide of sequence number 88; The first ESR1 primer of Sequence ID No. 65; The second ESR1 in sequence number 66; ESR1 competitive polynucleotide of sequence number 89; The first GSTM1 primer of sequence number 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 90; PGR primer No. 1 of Sequence ID No. 73; The second PGR of sequence number 74; PGR competing polynucleotide of sequence number 91; SCUBE2 primer No. 1 of Sequence ID No. 77; SCUBE2, the second element of sequence number 78; SCUBE2 competitive polynucleotide of sequence number 92; and Includes or comprises a universal estrogen module probe oligonucleotide of SEQ ID NO: 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of Sequence ID No. 57; The second BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 100; The first BCL2 primer of Sequence ID No. 61; The second BCL2 of sequence number 62; BCL2 competitive polynucleotide of sequence number 97; The first ESR1 primer of Sequence ID No. 65; The second ESR1 in sequence number 66; ESR1 competitive polynucleotide of sequence number 94; The first GSTM1 primer of sequence number 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 99; PGR primer No. 1 of Sequence ID No. 73; The second PGR of sequence number 74; PGR competing polynucleotide of sequence number 96; SCUBE2 primer No. 1 of Sequence ID No. 77; SCUBE2, the second element of sequence number 78; SCUBE2 competitive polynucleotide of sequence number 98; and The present invention comprises or consists of universal estrogen module probe oligonucleotides of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules.

[0113] In some embodiments, the kit is The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with Housekeeping Fluorescent Label. The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and Includes all universal estrogen module probe oligonucleotides.

[0114] As will be understood, the results and / or readouts of the methods implemented herein can be interpreted using algorithms, such as those suitable for the analysis of PCR data, qPCR data, etc. Such algorithms are known to those skilled in the art. As will be understood, algorithms can be used to calculate recurrence scores (RS) as disclosed herein.

[0115] The algorithms described herein may be performed on a computer.

[0116] In a further embodiment, an apparatus is provided, the apparatus is At least one processor, and It includes at least one memory containing computer program code for one or more programs, The device is configured to use at least one memory and computer program code to cause the device to implement any algorithm disclosed herein, using at least one processor.

[0117] A corresponding computer program for performing one or more steps of any algorithm disclosed herein is also within the scope of this disclosure and is encompassed by one or more of the embodiments described herein.

[0118] One or more computer programs, when executed on a computer, can cause the computer to configure any device disclosed herein or to execute any algorithm disclosed herein. One or more computer programs may be software implementations, and the computer may be considered any suitable hardware, including, in non-limiting examples, a digital signal processor, a microcontroller, and an implementation in read-only memory (ROM), erasable programmable read-only memory (EPROM), or electronically erasable programmable read-only memory (EEPROM). The software may also be an assembly program.

[0119] One or more computer programs may be provided on a computer-readable medium, which may be a physical computer-readable medium such as a disk or memory device, or they may be embodied as transient signals. Such transient signals may be network downloads, including internet downloads.

[0120] As seen herein, any list or discussion of previously published literature should not necessarily be construed as an endorsement that such literature is part of the latest technology or common general knowledge.

[0121] Preferred embodiments and options relating to a given aspect, feature, or parameter of the present invention should be deemed to be as disclosed in combination with all other aspects, features, and parameters of the present invention, unless otherwise indicated in the context.

[0122] The array of the present disclosure

[0123] [Table 3-1]

[0124] [Table 3-2]

[0125] [Table 3-3]

[0126] [Table 3-4]

[0127] [Table 3-5]

[0128] [Table 3-6]

[0129] [Table 3-7]

[0130] [Table 3-8]

[0131] Table 3-9

[0132] Table 3-10

[0133] Table 4-1

[0134] Table 4-2

[0135] Table 4-3

[0136] Table 4-4

[0137] Table 4-5

[0138] Table 4-6

[0139] Table 4-7

[0140] Table 4-8

[0141] Table 4-9 [Brief explanation of the drawing]

[0142] [Figure 1] a) The normalized emission spectra of a series of fluorophores provide a criterion for selecting potential candidates for multiplexing. b) Identify the optimal reporter for the quintuple assay in Rotor-Gene® Q by comparing the spectrum to the instrument's filter specifications. c) Bleed the signal from CAL Fluor Red 610 (red trace) to channel detection Quasar 670 (blue trace). Then remove crosstalk using software settings. [Figure 2] (A) A single competitive amplification unit consists of a native target (cDNA derived from mRNA), a synthetic competitive polynucleotide, a set of primers shared by both the native target and the competitive polynucleotide, and one or more probes. Each probe hybridizes to either the native target or the competitor, but not both, and each probe is labeled with a distinct fluorophore (here, FAM and HEX). The figure on the right shows a simplified representation of the structure of a competitive unit: squares represent the native target, circles represent the competitor, small dots represent the set of primers, and shaded areas represent any probe and each of their fluorophores. (B) A reaction module is a collection of competitive amplification units, each specific to a different native target, all utilizing probes that have the same fluorophore. For example, a given reaction module may contain a single probe sequence having the FAM fluorophore, or multiple different probe sequences, each having the FAM fluorophore. A single probe sequence may hybridize to only one competitor, or it may be designed to hybridize to multiple competitors. A given reaction module may contain probes specific to a competing sequence, a natural target, or a mixture of both. Those skilled in the art will understand that the fluorophores in a given module can be modified without significantly altering their behavior. [Figure 3]Assay configuration X. An assay configuration according to the method disclosed herein consists of multiple reactions, each carried out in a separate qPCR well. Each reaction consists of one or more modules. Furthermore, the reactions may optionally include nonspecific fluorescent dyes such as SYBR Green dye, EvaGreen dye, EvaGreen dye Plus, EvaRuby dye, or similar, which exhibit increased fluorescence in the presence of any double-stranded DNA, regardless of sequence. If a given reaction consists of a single module, the probe in that module may utilize any fluorophore known in the art, provided that it is compatible with any nonspecific fluorescent dye present. If the reaction consists of multiple modules, each module utilizes a separate fluorophore. These may be any fluorophore known in the art, provided that they are all compatible with each other and with any nonspecific fluorescent dye present. It will be understood by those skilled in the art that "compatible" means any two dyes that show limited overlap in excitation and / or emission spectra, but individually show significant overlap with the excitation and emission spectra of a given qPCR instrument. [Figure 4-1] Modules A, B, and C are shown. Those skilled in the art will recognize that many alternative permutations exist to produce configurations involving three and four reactions, and that the selection of specific / nonspecific dyes (EvaGreen dye, EvaRuby dye, or similar) relates only to the selection of fluorophores for a given module and not to the exemplary module targets shown herein. For example, configuration F may be modified such that the F4 reaction utilizes the FAM fluorophore for the estrogen+ module and contains the EvaRuby dye, while the F3 reaction utilizes the VIC fluorophore for the HER2 module and contains the EvaGreen dye. Note that in the illustrated configurations, the fact that two modules in different reactions have the same shading does not mean that they must share the same fluorophore. The selection of fluorophores in different reactions is independent of each other. [Figure 4-2]Modules D, E, and F are shown. Those skilled in the art will recognize that many alternative permutations exist to produce configurations involving three and four reactions, and that the selection of specific / nonspecific dyes (EvaGreen dye, EvaRuby dye, or similar) relates only to the selection of fluorophores for a given module and not to the exemplary module targets shown herein. For example, configuration F may be modified such that the F4 reaction utilizes the FAM fluorophore for the estrogen+ module and contains the EvaRuby dye, while the F3 reaction utilizes the VIC fluorophore for the HER2 module and contains the EvaGreen dye. Note that in the illustrated configurations, the fact that two modules in different reactions have the same shading does not mean that they must share the same fluorophore. The selection of fluorophores in different reactions is independent of each other. [Figure 5-1] Exemplary assay configurations A and B are shown. One exemplary embodiment consists of assay configuration B in Figure 5, which is demonstrated by the following data and examples, where: · Reaction B1 contains the housekeeping module consisting of SEQ ID NOs: 1-16, the fluorophore is VIC, and the reactant contains EvaGreen dye Plus; · Reaction B2 contains the growth module consisting of SEQ ID NOs: 29-48, the fluorophore is FAM; · Reaction B3 contains the HER2 module consisting of SEQ ID NOs: 49-56, the fluorophore is JUN; · Reaction B4 contains the estrogen+ module consisting of SEQ ID NOs: 57-80, the fluorophore is VIC; · Reaction B5 contains the wetting+ module consisting of SEQ ID NOs: 17-28, the fluorophore is ABY. [Figure 5-2] Exemplary assay configurations C and D are shown. [Figure 5-3] Exemplary assay configurations E and F are shown. [Figure 5-4] Exemplary assay configurations G and H are shown. [Figure 6-1]This shows the iterative optimization of primer sequences for a set of 32 primers (only for genes with high information content). (A) Evolution of the Pareto front - showing the best (highest) CT observed for any reaction containing a given number of primer groups over time. (B) Box plot showing the distribution of primer dimer CT values ​​for different reaction primer counts. [Figure 6-2] This shows iterative optimization of primer sequences for a set of 32 primers (high-information genes only). (C) Empirical cumulative density function (eCDF) for the largest set within each subset only. Horizontal or vertical dashed lines indicate an acceptable primer dimer CT threshold of 32.5. In total, 123 iterations were performed with a total of 47,232 reactions to identify the optimal set of sequences for each subset. Of the 631 different combinations of 32 high-information gene primer candidate sequences, only one (0.16%) had an acceptable CT value (greater than 32.5). [Figure 7-1] This shows the repeat optimization of primer sequences for a set of 42 primers (all genes). (A) Evolution of the Pareto front - shows the best (highest) CT observed for any reaction containing a given number of primer groups over time. (B) Box plot showing the distribution of primer dimer CT for different reaction primer counts. [Figure 7-2] The iterative optimization of primer sequences for a set of 42 primers (all genes) is shown. (C) Empirical cumulative density function (eCDF) for the largest set within each subset only. Horizontal or vertical dashed lines indicate an acceptable primer dimer CT threshold of 32.5. In total, 123 iterations were performed for a total of 47,232 reactions to identify the optimal set of sequences for each subset. Of the 588 different combinations of 42 primer candidate sequences for all genes in the assay, only one (0.16%) had an acceptable CT value (greater than 32.5). [Figure 8-1]Optimization of component concentrations for each module. This invention required careful optimization of the concentrations of each synthetic DNA component (primer group, probe, and competitor). The inventors first screened 177 different configurations for the HK module, as well as 499 different configurations for each of the FAM, VIC, ABY, and JUN modules. For each candidate configuration of each module, the inventors tested six simulated patient samples and measured the correlation between the predicted and observed signals. (A) The majority of the configurations showed insufficient (low) correlation (top row). Figure 8B shows the selection of 24 most promising configurations for each module, and this subset was tested against 60 simulated patient samples. Despite such expert selection, many of these still showed insufficient correlation even in this higher-resolution experiment. [Figure 8-2] Optimization of component concentrations for each module. This invention required careful optimization of the concentrations of each synthetic DNA component (primer group, probe, and competitor). The inventors first screened 177 different configurations for the HK module, and 499 different configurations for each of the FAM, VIC, ABY, and JUN modules. For each candidate configuration of each module, the inventors tested six simulated patient samples and measured the correlation between the predicted and observed signals. (B) For each module, 24 of the most promising configurations were selected, and this subset was tested against 60 simulated patient samples. Despite such expert selection, many of these showed insufficient correlation even in this higher-resolution experiment. [Figure 9]The OncoSignatur Breast assay was performed. Each assay module was run in a separate reaction (five in total: HK, FAM, VIC, ABY, JUN). Each module consisted of a set of primers for the relevant mRNA target ("wild-type amplicon"), a competing sequence, and a fluorescent probe specific to the competing sequence. (A) The correlation between the predicted score from each module (x axis) and the score observed from our assay for each simulated patient (individual dot) (y axis) is shown. The R value represents the Pearson correlation coefficient between the expected and observed values. Since the correlation is highly sensitive to outliers, the worst 10% of the data points (6) were excluded for each module. The inventors then used a modified Bayesian linear regression method to convert their five module measurements into a standardized "scale-free relapse score" (RSu) that assigns outliers from a standard normal distribution. (B) This was converted into a final relapse score using the OTDX patent method. These results demonstrate that the OncoSignatur Breast assay faithfully reproduces the highly correlated Oncotype DX relapse score. [Figure 10] Readout information from a representative sample. In an exemplary configuration, the present invention provides clearly distinguishable fluorescence signals from each module. Here, the signals obtained from a representative synthetic sample are shown. These signals are then processed by extracting the final fluorescence intensity at the end of the reaction (indicated here by dots). For comparison, the expected readout information from each module is shown as a horizontal dashed line. It is clear that the assay produces the expected signals from each module. [Figure 11-1]Six patient samples were tested using one embodiment of the housekeeping module. The samples consisted of formalin-fixed and paraffin-embedded (FFPE) excised mammary tumor tissue, from which RNA was extracted from three 5 μm sections. Total RNA was quantified from each sample, and the samples were then diluted to the indicated concentrations. Each reaction consisted of 1 μL of sample, 1 μL of 5× housekeeping module (containing EvaGreen dye), 1.25 μL of 4× one-step RT-qPCR master mix, and 1.75 μL of water. For reverse transcriptase conversion of RNA to DNA, the samples were incubated in a thermocycler at 53°C for 10 minutes, followed by incubation at 95°C for 2 minutes, and finally 50 cycles of 3 seconds at 95°C and 30 seconds at 60°C. The resulting fluorescence was normalized, background was subtracted, and the average fluorescence intensity at cycles 48, 49, and 50 was used as the housekeeping signal. This signal showed a strong dependence on sample dilution and a strong correlation with the cycle threshold of the EvaGreen dye signal, both indicating that the housekeeping signal is an accurate representation of the geometric mean CT values ​​of five housekeeping gene transcripts in patient samples. [Figure 11-2]Six patient samples were tested using one embodiment of the housekeeping module. The samples consisted of formalin-fixed and paraffin-embedded (FFPE) excised mammary tumor tissue, from which RNA was extracted from three 5 μm sections. Total RNA was quantified from each sample, and the samples were then diluted to the indicated concentrations. Each reaction consisted of 1 μL of sample, 1 μL of 5× housekeeping module (containing EvaGreen dye), 1.25 μL of 4× one-step RT-qPCR master mix, and 1.75 μL of water. For reverse transcriptase conversion of RNA to DNA, the samples were incubated in a thermocycler at 53°C for 10 minutes, followed by incubation at 95°C for 2 minutes, and finally 50 cycles of 3 seconds at 95°C and 30 seconds at 60°C. The resulting fluorescence was normalized, background was subtracted, and the average fluorescence intensity at cycles 48, 49, and 50 was used as the housekeeping signal. This signal showed a strong dependence on sample dilution and a strong correlation with the cycle threshold of the EvaGreen dye signal, both indicating that the housekeeping signal is an accurate representation of the geometric mean CT values ​​of five housekeeping gene transcripts in patient samples. [Figure 12] The OSB constructed with PCRchitectur (described in international application PCT / GB2021 / 052594, published as international publication 2022 / 074392(A1)) circumvents the conventional optical limitations of PCR multiplexing. While conventional reliance on CT limits a single fluorescence optical channel to examining only a single gene, PCRchitectur utilizes endpoint fluorescence as a highly informative signal property. This makes it possible to examine multiple genes within a single light channel. PCRchitectur uses competitive amplification to convert the concentration of a given target amplicon into fluorescence intensity (endpoint signal) at the end of the reaction. Unlike CT, the endpoint signal is additive: each signal from different genes examined within the same light channel is linearly combined to produce the sum of the observed signal intensities. [Figure 13-1]The OSB consists of five modules, each comprising a competitive amplification pair corresponding to a specific gene. Each module utilizes only a single optical channel, and modules can reside in a single reaction well or in separate wells (i.e., all competitive pairs for module 1 in well 1, all pairs for module 2 in well 2, etc., but in all cases, only a single optical channel is used for each module). When testing patient samples, each competitive pair produces a specific endpoint signal intensity. Therefore, the fluorescence observed from each module is the sum of the signals from its component amplification pairs. These per-module endpoint signals can then be algorithmically combined to generate a risk score for each patient. For example, high-risk patients with a score above a certain threshold are at high risk of recurrence within the next 10 years. The arrows in the "High-Risk Patient" and "Low-Risk Patient" boxes correspond to data from labeled modules 1-4. [Figure 13-2] The OSB consists of five modules, each comprising a competitive amplification pair corresponding to a specific gene. Each module utilizes only a single optical channel, and modules can reside in a single reaction well or in separate wells (i.e., all competitive pairs for module 1 in well 1, all pairs for module 2 in well 2, etc., but in all cases, only a single optical channel is used for each module). When testing patient samples, each competitive pair produces a specific endpoint signal intensity. Therefore, the fluorescence observed from each module is the sum of the signals from its component amplification pairs. These per-module endpoint signals can then be algorithmically combined to generate a risk score for each patient. For example, high-risk patients with a score above a certain threshold are at high risk of recurrence within the next 10 years. The arrows in the "High-Risk Patient" and "Low-Risk Patient" boxes correspond to data from labeled modules 1-4. [Figure 14]A) The OncoSignatur Breast prototype achieved a 90% agreement with Oncotype DX (89 out of 99 samples). B) Extrapolating these results to the general population provides estimates of what results might be obtained from a large, representative population. [Figure 15] Further details are provided regarding the values ​​for each sample, the trade-off between sensitivity and specificity, and the trade-off between high and low predictive values. [Figure 16] The algorithm and specific values ​​used in the study of Example 5 are shown below. Here, "Prol" refers to the endpoint signal from the Prol module for a given sample divided by the signal from its respective control reaction (reaction with a competitor, primer group, and probe only, which shows the maximum possible signal from the Prol module). Estp, Invp, Her2, and Hskp are defined similarly. All other values ​​are provided in the chart on the following slide. [Examples]

[0143] Example 1: Optimization of primer sequence As described elsewhere in this specification, as well as in the drawings and descriptions of the drawings, the method of the present invention may be carried out in up to five separate reactions or in a single reaction. A single reaction for the amplification of all high-information modules (growth, invasion, HER2, and estrogen) requires amplification of 16 target polynucleotides, which means that 32 primers are present in the reaction. A single reaction for the amplification of all modules (housekeeping, growth, invasion, HER2, and estrogen) requires amplification of 21 target polynucleotides, which means that 42 primers are present in the reaction.

[0144] This invention required careful optimization of primer sequences to avoid harmful primer dimer formation. Primer sequences were optimized using high-throughput experiments and sophisticated computer simulation techniques. Figure 6A shows the evolution of the Pareto front, the best (highest) CT observed for any reaction containing a given number of primer groups over time. Each replicate consisted of testing different combinations of candidate primer sequences in each well of a 384 qPCR plate (using qPCR master mix and EvaGreen dye), observing the signal obtained over 50 PCR cycles, and determining the CT of the reaction. In the ideal scenario, no primer dimers are formed, and no detectable signal is produced (recorded as CT 50). Figure 6B shows a box plot illustrating the distribution of primer dimer CT for different reaction primer counts, and Figure 6C shows the empirical cumulative density function (eCDF) for the largest set within each subset. Horizontal or vertical dashed lines indicate an acceptable primer dimer CT threshold of 32.5. In total, 123 replicates were performed with a total of 47,232 reactions to identify the optimal sequence set for each subset. Of the 631 different combinations of primer candidate sequences for 32 high-information genes, only one (0.16%) had an acceptable CT value (greater than 32.5).

[0145] Figure 7 shows the primer sequence repeat optimization for a 42-primer (whole gene) set. Figure 7A shows the evolution of the Pareto front—the evolution over time of the best (highest) CT observed for any reaction containing a given number of primer groups. Each repeat consisted of testing different combinations of candidate primer sequences in each well of a 384 qPCR plate (using qPCR master mix and EvaGreen dye), observing the signal obtained over 50 PCR cycles, and determining the CT of the reaction. In an ideal scenario, no primer dimers are formed, and no detectable signal is produced (recorded as CT 50). Figure 7B shows a box plot illustrating the distribution of primer dimer CT for different reaction primer counts, and Figure 7C shows the empirical cumulative density function (eCDF) for the largest set within each subset. Horizontal or vertical dashed lines indicate an acceptable primer dimer CT threshold of 32.5. In total, 123 repeats were performed for a total of 47,232 reactions to identify the optimal sequence set for each subset. Of the 588 different combinations of 42 primer candidate sequences for all genes in the assay, only one (0.16%) had an acceptable CT value (greater than 32.5). The sequences of these primers are described elsewhere in this specification, for example, in Table 1.

[0146] Example 2: Optimization of component concentrations in each module This invention required careful optimization of the concentrations of each synthetic DNA component (primer group, probe, and competitor). First, 177 different configurations of the HK module, as well as 499 different configurations each of the FAM, VIC, ABY, and JUN modules, were screened. For each candidate configuration in each module, six simulated patient samples were tested, and the correlation between the predicted and observed signals was measured. Figure 8A shows that the majority of the configurations showed insufficient (low) correlation. Twenty-four of the most promising configurations were selected for each module and tested against 60 simulated patient samples. Despite such expert selection, many of these still showed insufficient correlation even in this higher-resolution experiment (Figure 8B).

[0147] Example 3: Implementation of the assay of the present invention (OncoSignatur Breast assay) As described elsewhere in this specification, for example in Figure 5, there are many different permutations in which the assays and methods of the present invention can be carried out. Figure 9 shows the data obtained when the assay was carried out according to the embodiment described in configuration B of Figure 5. Each assay module is performed in a separate reaction (five in total: HK, FAM, VIC, ABY, JUN). Each module consists of a set of primers for the relevant mRNA target ("wild-type amplicon"), a competing sequence, and a fluorescent probe specific to the competing sequence. [Note that there is no probe specific to the "wild-type amplicon" (referred to herein as the "target amplicon"). However, as will be apparent to those skilled in the art, in some embodiments of the present invention, the probe may be specific to the target amplicon rather than the competing amplicon.]

[0148] After subtracting the background, the readout information from each module consists of the fluorescence intensity from the associated fluorophore after 50 cycles of qPCR amplification. Figure 10 shows the readout information for a representative sample. In an exemplary configuration, the present invention provides clearly distinguishable fluorescence signals from each module. Here, the signals obtained from a representative synthetic sample are shown. These signals are then processed by extracting the final fluorescence intensity at the end of the reaction (indicated here by dots). For comparison, the expected readout information for each module is shown as a horizontal dashed line. It is clear that the assay produces the expected signals from each module.

[0149] The inventors tested these modules on 60 "simulated patients" (a mixture of synthetic DNA in a buffer designed to statistically mimic realistic mRNA levels in actual patients). Figure 9A shows the correlation between the predicted score from each module (x-axis) and the score observed from the inventors' assay for each simulated patient (individual dots) (y-axis). The R-value represents the Pearson correlation coefficient between the expected and observed values. Because the correlation is highly sensitive to outliers, the worst 10% of data points (6) were excluded for each module. The inventors then used a modified Bayesian linear regression method to convert the inventors' five module measurements into a standardized "scale-free relapse score" (RSu) that assigns outliers from a standard normal distribution. This was then converted into a final relapse score using the OTDX patent method (Figure 9B).

[0150] The final formula is as follows: RSuZ=max(β_FAM * (HK-FAM),-γ_FAM)-β_VIC * (HK-VIC)+β_ABY * (HK-ABY)+max(β_JUN * (HK-JUN),-γ_JUN) RSu=RSuZ * 1.47 + 9.98 RS=min(max(20 * (RSu-6.7),0),100) Here, β_FAM=0.35, β_VIC=0.42, β_ABY=0.17, β_JUN=0.46, γ_FAM=1.14, and γ_JUN=0.76, where HK, FAM, VIC, ABY, and JUN are standardized endpoint fluorescence intensities from the relevant modules.

[0151] These results demonstrate that the OncoSignatur Breast assay faithfully reproduces the highly correlated Oncotype DX relapse score.

[0152] Example 4: The housekeeping module accurately reports the sample volume. Refer to Figure 11. Six patient samples were tested using one embodiment of the housekeeping module. The samples consisted of formalin-fixed and paraffin-embedded (FFPE) excised mammary tumor tissue, from which RNA was extracted from three 5 μm sections. Total RNA was quantified from each sample, and the samples were then diluted to the indicated concentrations. Each reaction consisted of 1 μL of sample, 1 μL of 5× housekeeping module (containing EvaGreen dye), 1.25 μL of 4× one-step RT-qPCR master mix, and 1.75 μL of water. For reverse transcriptase conversion of RNA to DNA, the samples were incubated in a thermocycler at 53°C for 10 minutes, followed by incubation at 95°C for 2 minutes, and finally 50 cycles of 3 seconds at 95°C and 30 seconds at 60°C. The resulting fluorescence was normalized, background was subtracted, and the average fluorescence intensity at cycles 48, 49, and 50 was used as the housekeeping signal. This signal showed a strong dependence on sample dilution and a strong correlation with the cycle threshold of the EvaGreen signal, both indicating that the housekeeping signal is an accurate representation of the geometric mean CT values ​​of five housekeeping gene transcripts in patient samples.

[0153] Example 5: Summary of the OncoSignatur Breast Preliminary Clinical Trial overview: • The initial prototype of OncoSignatur Breast achieved a 90% match with the gold standard, OncoType DX. • Predictive accuracy for low-risk patients was 94%, demonstrating a strong safety profile. The prototype test kit demonstrated flexibility in international transport and operator and equipment variability. Extrapolating these results to the score distribution in the general population suggests that very large representative studies already achieve a good agreement rate of 88–91% compared to existing commercially available test kits. A detailed analysis of response behavior suggests specific optimization steps that are predicted to achieve an overall agreement of 95% in a typical population.

[0154] background The inventors have developed OncoSignatur Breast (OSB, summarized in Figures 12 and 13), a distributed test kit for postoperative identification of early-stage breast cancer patients at low risk of recurrence. This test analyzes the expression of 21 genes in the patient's tumor to generate a score from 0 to 100, where a higher value indicates a higher risk of recurrence and suggests that the patient should be prescribed chemotherapy in addition to endocrine therapy, while a lower value indicates a lower risk of recurrence and suggests that chemotherapy can be safely avoided in favor of endocrine therapy alone. To provide a preliminary evaluation of test performance in a distributed environment and guide future development, Signature Biosciences partnered with Unidad de Genomica (Madrid, Spain) to conduct an exploratory, open-label, retrospective study on the performance of an early-stage prototype of OncoSignatur Breast. As a substitute for actual results, we compared the OncoSignatur Breast score with historical scores from the well-known existing trial, Oncotype DX, and investigated any differences, particularly in how the two trials classified patients as high-risk and low-risk.

[0155] Research timeline This study involved the analysis of breast tumor specimens from 99 patients collected between 2012 and 2015 and stored in FFPE blocks. The patient FFPE tumor specimens were initially collected as part of an unpublished CONCORD clinical trial. In 2016, sections were sent to Exact Sciences for oncotype DX scoring, and in parallel, RNA was isolated using the Invitrogen RecoverAll kit for downstream RT-qPCR analysis. After completion of the study objectives, residual RNA was sent to a clinical partner at La Paz Hospital in Madrid and stored at -80°C for further research use. Access to previously extracted RNA was more convenient than access to the original FFPE samples; therefore, the inventors chose to use the former to mitigate initial risks in this study. Future studies, including the inventors' training, validation, and verification work, as outlined below, will be performed directly on FFPE samples under strict, forward-looking, defined protocols.

[0156] The inventors assembled the kit components for OSB at their laboratory in New Haven, Connecticut, and loaded them into 20 384-well qPCR plates on dry ice before shipping them to Madrid, Spain. The plates were frozen at -20°C and used within two weeks.

[0157] The prototype test kit was performed by non-affiliated laboratory technicians after brief training by Signatur Biosciences employees. The collected samples were first examined for quality and concentration using an Agilent Tapestation, which revealed that the samples had lost significant concentration during storage (median loss 70%, CI 57–86%), and the samples generally exhibited low quality (median RIN: 2.3). The RNA completeness number (RIN) ranges from 1 to 10, where 10 represents ideal RNA quality and 1 means the most degraded. Sample aliquots were diluted to 1 ng / uL and then combined with the master mix and water to form the pre-packed kit components. The plates also contained individual primer pairs for direct quantification of the expression of each gene in the signature. The plates were run on a Quantstudio 12K within 20 minutes of assembly, and the data files were returned to Signature Biosciences for analysis (note that the tests were developed on Quantstudio 6 and 7 Pro DX).

[0158] result OncoSignatur Breast showed a 90% (95%, CI 83-95%: confidence intervals for observation were calculated using one-out cross-validation and Bayesian β-binomial regression) agreement with the Oncotype DX relapse score (RS). Sensitivity in low-risk patients was 94% (88-98%) and specificity was 61% (35-84%) (standard treatment without prognostic trials is to prescribe chemoendocrine therapy; therefore, in classical terms, low-risk patients are considered test-positive and high-risk patients are test-negative). To understand how these results translate to a very large representative sample without further refinement, the inventors estimated the distribution of recurrence scores in the general population from three published studies encompassing nearly 700,000 patients (Blohmer et al., "Population sizes of patients (pts) with node negative (N0), HR+, HER2-primary breast cancer (BC), using standard and TAILORx 21-gene recurrence score (RS) cut-off values ​​(COV)", ESMO, 2017; Sparano et al., "Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer", New England Journal of Medicine 379:111-121 (2018); Mariotto et al., "Expected Monetary Impact of Oncotype DX Score-Concordant Systemic Breast Cancer Therapy Based on the TAILORx Trial", J Natl Cancer). Inst, 112:154-160 (2019).Next, the inventors estimated the predictive value of their assay for different RS values ​​(with uncertainty – confidence intervals for extrapolated performance were derived from the 95% highest density interval estimate of Bayesian noise in observations), and extrapolated their findings by conditioning the estimated population distribution, achieving an agreement of 88–91%, sensitivity of 92–95%, specificity of 70–73%, predictive value of approximately 94% for low-risk patients, and predictive value of 66–77% for high-risk patients. Regarding sensitivity versus specificity, the AUROC from the inventors' observations was 0.85, compared to 0.92–0.95 from the inventors' population estimates. Regarding high-risk versus low-risk predictive values, the AUROC from the inventors' observations was 0.93, compared to 0.95–0.97 from the inventors' population estimates. Cross-sectional analysis showed no significant differences in performance based on lymph node metastasis status or menopausal status. Please refer to Figures 14, 15, and 16.

[0159] After establishing the current performance of the prototype system, we desired to better understand potential pathways for improvement. We recalculated recurrence scores from measured CTs of individual genes, enabling us to independently evaluate the performance of each of the five submodules within OncoSignatur Breast (these correspond to the published housekeeping, proliferation, estrogen, invasion, and HER2 groups within the Oncotype DX signature, with the remaining negatively and positively correlated genes grouped together with the estrogen and invasion groups, respectively). Next, we simulated improvements to the estimates for each of these modules toward their respective ideals, allowing us to predict the impact of various optimization efforts.

[0160] The oligonucleotides used in this study are as follows: Amplification of the housekeeping module using the following oligonucleotides: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competitive polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competing polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competitive polynucleotide of SEQ ID NO: 106; and Universal Housekeeping Module Probe Oligonucleotide of Sequence ID No. 102, labeled with Housekeeping Fluorescent Label. and The infiltration module is amplified using the following oligonucleotides: The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; and The amplification of the proliferation module is performed using the following oligonucleotides: The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and SEQ ID NO: 45 (first MYBL2 primer); SEQ ID NO: 46 (second MYBL2 primer); MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; and The Her2 module is amplified using the following oligonucleotides: The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; and The estrogen module is amplified using the following oligonucleotides: The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 96; SEQ ID NO: 77 (First SCUBE2 primer); SEQ ID NO: 78 (Second SCUBE2); SEQ ID NO: 98 (SCUBE2 competitive polynucleotide); and Universal estrogen module probe oligonucleotide.

[0161] Consideration This study validated several proposals regarding the significant value of OncoSignatur Breast. Firstly, the observed overall agreement and predicted improvements indicate that OSB is well on track to achieve substantially equivalent performance to the current gold standard. The lack of significant differences in performance between premenopausal and postmenopausal patients, and between lymph node metastasis-positive and lymph node metastasis-negative patients, means that OncoSignatur Breast is unbiased and therefore broadly applicable. Extrapolation to the general population suggests that these results would hold true, and possibly even slightly improve, in large, representative studies. The remaining modules, excluding the housekeeping module, consist of 2, 3, 5, and 6 genes, and there are no significant qualitative differences among them; i.e., there is no reason to anticipate substantially greater difficulty in tuning their behavior to the housekeeping level. These predicted improvements to module fidelity are readily achievable through iterative optimization of transducer concentrations.

[0162] Secondly, this study demonstrated that OncoSignatur Breast performed well in the hands of typical real-world users, even in a dispersed environment after international transport, using equipment different from that used in development. This is evidence of a robust control system that effectively mitigates inter-well, inter-device, inter-operator, and environmental variability. As this was an early-stage prototype, the company had not yet performed even basic optimizations for manufacturing and distribution. The degree of performance improvement expected to result from increased robustness suggests prioritizing efforts toward manufacturing reproducibility, selecting various plastics and additives for storage stability, and ensuring that shipping procedures properly maintain the appropriate environment.

[0163] Robust predictive values ​​for low-risk patients already support a strong safety profile: the probability of misclassifying a high-risk patient as low-risk is approximately 1 in 20. This is important because misclassification from high-risk to low-risk and misclassification from low-risk to high-risk are not equivalent in their impact: misclassification from high-risk to low-risk means that patients who should receive chemotherapy are advised to postpone it, increasing the risk of avoidable death, while misclassification from low-risk to high-risk results in unnecessary chemotherapy and impairs the patient's quality of life, but is less likely to affect long-term survival; therefore, misclassification from high-risk to low-risk is clinically worse than misclassification from low-risk to high-risk. The results demonstrate very high reliability in low-risk classification with the current configuration of OncoSignatur Breast.

[0164] The precise trade-off between the two types of error is an algorithmic choice, not a strict outcome of the kit design. Therefore, any particular value of one metric, such as low-risk predictors, can be chosen at the expense of complementary metrics. For example, the OncoSignatur Breast algorithm could be configured to show 99% low-risk predictors (i.e., 1 in 100 patients classified as low-risk is actually high-risk) in the current kit design. However, this configuration would reduce high-risk predictors to 31-43%, lowering the overall agreement to 61-86%. An acceptable trade-off is a clinical judgment determined through conversations with clinicians and regulatory bodies prior to the pre-validation algorithm freeze. In any case, a high AUROC well above 0.90 for predictors indicates strong overall performance, as well as the ability to adapt to a wide range of clinical and regulatory requirements.

[0165] Please refer to Figure 14 for the data.

[0166] With a 90% agreement rate, OncoSignatur Breast is ahead of most of its competitors. For comparison, the Veracyte Prosigna test showed an agreement rate of 80–97% with Oncotype DX, while Endopredict and Mammaprint only achieved 79–82% and 75–81%, respectively (Varga, et al., "Summary of head-to-head comparisons of patient risk classifications by the 21-gene Recurrence Score® (RS) assay and other genomic assays for early breast cancer." International Journal of Cancer, 145:882-893 (2019)). Meanwhile, previous FDA decisions indicate that OncoSignatur Breast's predictive accuracy is already close to acceptable. In their 510(k) submission, Veracyte argued that the Prosigna test predicted 94.4–97.9% for 10-year distant recurrence-free survival (i.e., low-risk patients) for lymph node-positive patients and 88–97% for lymph node-negative patients, but only 11–22% and 19–31% for recurrence within that timeframe (see FDA 510(k) Premarket Notification K130010, https: / / www.accessdata.fda.gov / scripts / cdrh / cfdocs / cfpmn / pmn.cfm?ID=K130010, and https: / / www.accessdata.fda.gov / cdrh_docs / reviews / k130010.pdf).

[0167] [Table 5]

[0168] Appendix to Statistical Methods Threshold selection It is important to note that the primary performance metrics (agreement, sensitivity, specificity, and high-risk and low-risk predictions) are highly dependent on the selection of thresholds between scores considered "high-risk" and scores considered "low-risk."12 Compared to Oncotype DX classification, setting a lower threshold reduces misclassification from high-risk to low-risk, but at the cost of increasing misclassification from low-risk to high-risk. Conversely, setting a higher threshold results in the opposite trade-off, where overall agreement is maximized by selecting a threshold that equalizes the proportions of these two types of misclassifications. For simplicity, each analysis presented here selected a threshold that resulted in a 1:1 ratio of misclassifications.

[0169] For the purpose of simplifying clinical communication, one might instead choose to maintain a threshold similar to that of Oncotype DX 25, and instead systematically shift the score higher or lower. This is mathematically equivalent, but visualizing the threshold shift is easier, and therefore we show it here instead.

Claims

1. A method for amplifying multiple target polynucleotides in a sample, a) A step of providing a sample potentially containing a target polynucleotide, b) Providing a plurality of sets of target competitive amplification oligonucleotides, wherein each set of target competitive amplification oligonucleotides is designed to amplify a specific target polynucleotide, and each target to be amplified has a corresponding set of target competitive amplification oligonucleotides, and within each set: i) Competitive polynucleotides; ii) A first primer and a second primer that can hybridize to a specific target polynucleotide and a first competing polynucleotide of a set, wherein the hybridization of the first primer and the second primer to the specific target polynucleotide enables amplification of a portion of the specific target polynucleotide that generates a target amplicon, and the hybridization of the first primer and the second primer to the first competing polynucleotide enables amplification of the first competing polynucleotide or a portion of the first competing polynucleotide that generates a competing amplicon; iii) A probe oligonucleotide comprising a fluorescent label and capable of hybridizing to the first competing amplicon or the first target amplicon, The first competing polynucleotide in each set is designed to have a different amplification dynamic than that of the first target polynucleotide in each set, step, c) A step in initiating the primer extension reaction, At least two, i.e., portions of each of the target polynucleotides are amplified, resulting in the generation of at least two, i.e., multiple different target amplicons, corresponding to each specific target; Amplification occurs in each competitive polynucleotide or a portion of each competitive polynucleotide in each set of target competitive amplification oligonucleotides, resulting in the generation of at least two, i.e., multiple, different competitive amplicons. The plurality of target polynucleotides are cDNA molecules derived from first and second or more different mRNA polynucleotides present in the sample obtained from the subject, and the plurality of mRNA polynucleotides are the following genes: ACTB, GAPDH, GUSB, RPLP0, TFRC (these together constitute the housekeeping module); CD68, CTSV, MMP11 (these together constitute an infiltration module); AURKA, BIRC5, CCNB1, MKI67, MYBL2 (these together constitute a breeding module); ERBB2, GRB7 (these together constitute the HER2 module); and / or - Expressed from a group including or consisting of BAG1, BCL2, ESR1, GSTM1, PGR, SCUBE2 (these together constitute the estrogen module), and 1) The set of competitive amplification oligonucleotides for amplification of the housekeeping module is: i) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competitive polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competing polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competing polynucleotide of SEQ ID NO: 106; and a universal housekeeping module probe oligonucleotide of Sequence ID No. 102 labeled with a housekeeping fluorescent label; or ii) The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competing polynucleotide of SEQ ID NO: 3; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competing polynucleotide of SEQ ID NO: 6; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competing polynucleotide of SEQ ID NO: 9; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 12; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competing polynucleotide of SEQ ID NO: 15; and comprising or consisting of a universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label; 2) A set of competitive amplification oligonucleotides for amplification of the infiltration module is: i) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; or ii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 20; and the CD68 probe oligonucleotide of SEQ ID NO: 19, labeled with a fluorescent label for the infiltration module; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competing polynucleotide of SEQ ID NO: 24; and the CTSV probe oligonucleotide of SEQ ID NO: 23, labeled with a fluorescent label for infiltration modules; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 28; and the MMP11 probe oligonucleotide of SEQ ID NO: 27, labeled with a fluorescent label for infiltration modules; or iii) The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 81; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 82; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 83; and comprising or consisting of a universal infiltration module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for infiltration modules; 3) A set of competitive amplification oligonucleotides for amplification of the proliferation module, i) the first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; The MYBL2 competitive polynucleotide of sequence number 115; and comprising or consisting of a universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; or ii) The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and the AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for the proliferation module; The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and the BIRC5 probe oligonucleotide of SEQ ID NO: 35, labeled with a fluorescent label for the proliferation module; The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and the CCNB1 probe oligonucleotide of SEQ ID NO: 39, labeled with a fluorescent label for the proliferation module; The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and the MK167 probe oligonucleotide of SEQ ID NO: 43, labeled with a fluorescent label for the proliferation module; comprising or consisting of: the first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; the MYBL2 competitive polynucleotide of SEQ ID NO: 48; and the MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for the proliferation module; or iii) The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 40; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 44; and The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; The MYBL2 competitive polynucleotide of sequence number 48; and comprising or consisting of a universal proliferation module probe oligonucleotide of Sequence ID No. 85, labeled with a fluorescent label for proliferation modules; 4) A set of competitive amplification oligonucleotides for amplification of the HER2 module, i) the first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and A universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules, or comprising such oligonucleotides; or ii) the first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competing polynucleotide of SEQ ID NO: 52; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and A universal HER2 module probe oligonucleotide of Sequence ID No. 86, labeled with a fluorescent label for HER2 modules, or comprising such oligonucleotides; or iii) First ERBB2 primer of SEQ ID NO: 49; second ERBB2 of SEQ ID NO: 50; The ERBB2 competitive polynucleotide of SEQ ID NO: 52; and the ERBB2 probe oligonucleotide of SEQ ID NO: 51, labeled with a fluorescent label for the HER2 module; comprising or consisting of: the first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 56; and the GRB7 probe oligonucleotide of SEQ ID NO: 55, labeled with a fluorescent label for the HER2 module; ＼ And / or 5) A set of competitive amplification oligonucleotides for amplification of the estrogen module, i) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competing polynucleotide of SEQ ID NO: 96; The first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 98; and comprising or consisting of a universal estrogen module probe oligonucleotide of Sequence ID No. 95, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competing polynucleotide of SEQ ID NO: 87; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 88; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 89; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 90; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competing polynucleotide of SEQ ID NO: 91; The first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 92; and comprising or consisting of a universal estrogen module probe oligonucleotide of Sequence ID No. 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 60; and the BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for the estrogen module; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 64; and the BCL2 probe oligonucleotide of SEQ ID NO: 63, labeled with a fluorescent label for the estrogen module; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 68; and the ESR1 probe oligonucleotide of SEQ ID NO: 67, labeled with a fluorescent label for the estrogen module. The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 72; and the GSTM1 probe oligonucleotide of SEQ ID NO: 71, labeled with a fluorescent label for the estrogen module; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competitive polynucleotide of SEQ ID NO: 76; and the PGR-1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for the estrogen module; A method comprising or consisting of: a first SCUBE2 primer of SEQ ID NO: 77; a second SCUBE2 of SEQ ID NO: 78; a SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and a SCUBE2 probe oligonucleotide of SEQ ID NO: 79 labeled with a fluorescent label for estrogen modules.

2. The method according to claim 1, wherein the plurality of target polynucleotides are cDNA molecules derived from mRNA polynucleotides expressed from one or more modules.

3. The method according to claim 1 or 2, wherein the method comprises optionally amplifying the target polynucleotides of the housekeeping module, the invasion module, the proliferation module, the HER2 module, and / or the estrogen module in separate or the same reaction.

4. The method according to any one of claims 1 to 3, wherein the fluorescent labels for each module present in the amplification reaction have different but interchangeable fluorescent labels.

5. The method according to any one of claims 1 to 4, wherein the fluorescent label for the module is selected from ABY, FAM, JUN, and VIC.

6. The method according to any one of claims 1 to 5, wherein the sample obtained from the subject is a tumor specimen, or optionally a breast tumor biopsy.

7. The method according to any one of claims 1 to 6, wherein the amplification reaction is a PCR reaction.

8. The method according to any one of claims 1 to 7, wherein the amplification reaction is a PCR-based reaction and is carried out for a specific number of cycles, optionally at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, or 60 cycles.

9. The method according to claim 8, further comprising the step of determining the fluorescence signal intensity of each fluorescent label associated with a probe oligonucleotide present in the amplified reactant generated after a specific number of cycles.

10. The method according to any one of claims 1 to 9, wherein the fluorescence signal intensity of each fluorescent label associated with the probe oligonucleotide present in the amplified reactant is determined after 50 amplification cycles.

11. The method according to any one of claims 1 to 10, further comprising determining the endpoint fluorescence obtained after a specific number of cycles of the fluorescent label associated with the housekeeping module, the invasion module, the growth module, the HER2 module, and the estrogen module.

12. A method for generating a risk score for use in determining the postoperative risk of breast cancer recurrence, and / or for predicting a response to chemotherapy, comprising carrying out the method according to any one of claims 1 to 11.

13. The method described above is below: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competing polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competing polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competing polynucleotide of SEQ ID NO: 106; and amplification of a housekeeping module using oligonucleotides of the universal housekeeping module probe oligonucleotide of sequence number 102, which is labeled with a housekeeping fluorescent label. and below: The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Amplification of an infiltration module using oligonucleotides, using the universal infiltration module probe oligonucleotide of SEQ ID NO: 108, which is labeled with a fluorescent label for infiltration modules. and below: The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; The MYBL2 competitive polynucleotide of sequence number 115; and Amplification of a growth module using oligonucleotides with the universal growth module probe oligonucleotide of SEQ ID NO: 112, labeled with a fluorescent label for growth modules. and below: The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119 and Amplification of a HER2 module using oligonucleotides with the universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; and below: The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competing polynucleotide of SEQ ID NO: 96; The first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 98; and This includes the amplification of the estrogen module using the universal estrogen module probe oligonucleotide, The method according to any one of claims 1 to 12, wherein each module is optionally amplified by a reaction separate from the amplification of the other modules, thereby generating module-specific endpoint fluorescence.

14. The method described above is a) Amplification of the housekeeping module, which includes or comprises target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or comprising the following genes: ACTB, GADPH, GUSB, RPLP0, and TFRC, wherein the set of competitively amplified polynucleotides used for amplification is: The first ACTB primer of Sequence ID No. 1; The second ACTB primer of Sequence ID No. 2; ACTB competing polynucleotide of Sequence ID No. 3; The first GADPH primer of Sequence ID No. 4; The second GADPH primer of Sequence ID No. 5; GADPH competing polynucleotide of sequence number 6; The first GUSB primer of Sequence ID No. 7; The second GUSB primer of Sequence ID No. 8; GUS competitive polynucleotide of sequence number 9; The first RPLP0 primer of Sequence ID No. 10; The second RPLP0 primer of Sequence ID No. 11; RPLP0 competitive polynucleotide of sequence number 12; The first TRFC primer of Sequence ID No. 13; The second TRFC primer of Sequence ID No. 14; TRFC competitive polynucleotide of SEQ ID NO: 15; and Amplification of the housekeeping module comprising or consisting of a universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label; and b) Amplification of the infiltration module comprising or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group comprising or consisting of the following genes: CD68, CTSV, and MMP11, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 20; and CD68 probe oligonucleotide of Sequence ID No. 19, labeled with a fluorescent label for infiltration modules; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competitive polynucleotide of SEQ ID NO: 24; and CTSV probe oligonucleotide of Sequence ID No. 23, labeled with a fluorescent label for infiltration modules; and The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of SEQ ID NO: 28; and MMP11 probe oligonucleotide of sequence number 27, labeled with a fluorescent label for infiltration modules; or ii) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 81; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competing polynucleotide of sequence number 82; The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of sequence number 83; and Amplification of the infiltration module comprising or consisting of a universal infiltration module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for infiltration modules; and c) Amplification of the growth module comprising or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group comprising or consisting of the following genes: AURKA, BIRC5, CCNB1, MK167, and MYBL2, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 32; and AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for a growth module; The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 36; and BIRC5 probe oligonucleotide of SEQ ID NO: 35, labeled with a fluorescent label for a growth module; The first CCNB1 primer of Sequence ID No. 37; The second CCNB1 primer of Sequence ID No. 38; CCNB1 competitive polynucleotide of sequence number 40; and CCNB1 probe oligonucleotide of Sequence ID No. 39, labeled with a fluorescent label for a growth module; The first MK167 primer of Sequence ID No. 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of SEQ ID NO: 44; and MK167 probe oligonucleotide of sequence number 43, labeled with a fluorescent label for a growth module; The first MYBL2 primer of Sequence ID No. 45; MYBL2, the second element of sequence number 46; The MYBL2 competitive polynucleotide of sequence number 48; and The MYBL2 probe oligonucleotide of sequence number 47, labeled with a fluorescent label for the proliferation module, or comprising the above, or ii) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of Sequence ID No. 37; The second CCNB1 primer of Sequence ID No. 38; CCNB1 competitive polynucleotide of sequence number 40; and The first MK167 primer of Sequence ID No. 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of sequence number 44; and The first MYBL2 primer of Sequence ID No. 45; MYBL2, the second element of sequence number 46; The MYBL2 competitive polynucleotide of sequence number 48; and Amplification of the growth module comprising or consisting of a universal growth module probe oligonucleotide of Sequence ID No. 85, labeled with a fluorescent label for growth modules; and d) The following genes: amplification of a HER2 module containing or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group including or consisting of ERBB2 and GRB7, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; The ERBB2 competitive polynucleotide of SEQ ID NO: 52; and ERBB2 probe oligonucleotide of SEQ ID NO: 51, labeled with a fluorescent label for HER2 modules; The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of SEQ ID NO: 56; and It comprises or consists of a GRB7 probe oligonucleotide of Sequence ID No. 55, labeled with a fluorescent label for the HER2 module; or ii) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of sequence number 52; and The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of SEQ ID NO: 56; and Amplification of the proliferation module comprising or consisting of a universal HER2 module probe oligonucleotide of Sequence ID No. 86, labeled with a fluorescent label for the HER2 module; and e) Amplification of the estrogen module comprising or consisting of target polynucleotides, which are cDNA molecules derived from mRNA polynucleotides expressed from the group comprising or consisting of the following genes: BAG1, BCL2, ESR1, GSTM1, PGR, and SCUBE2, wherein the set of competitively amplified polynucleotides used for amplification is: i) The first BAG1 primer of Sequence ID No. 57; BAG1 of sequence number 58; BAG1 competitive polynucleotide of SEQ ID NO: 60; and BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for estrogen modules; The first BCL2 primer of SEQ ID NO: 61; The second BCL2 of sequence number 62; BCL2 competitive polynucleotide of SEQ ID NO: 64; and BCL2 probe oligonucleotide of SEQ ID NO: 63, labeled with a fluorescent label for estrogen modules; The first ESR1 primer of Sequence ID No. 65; The second ESR1 of sequence number 66; ESR1 competitive polynucleotide of SEQ ID NO: 68; and ESR1 probe oligonucleotide of SEQ ID NO: 67, labeled with a fluorescent label for estrogen modules; The first GSTM1 primer of Sequence ID No. 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of SEQ ID NO: 72; and GSTM1 probe oligonucleotide of SEQ ID NO: 71, labeled with a fluorescent label for estrogen modules; The first PGR primer of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of SEQ ID NO: 76; and PGR1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for estrogen modules; The first SCUBE2 primer of Sequence ID No. 77; SCUBE2 of sequence number 78; SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and comprising or consisting of SCUBE2 probe oligonucleotides of SEQ ID NO: 79, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of Sequence ID No. 57; BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 87; The first BCL2 primer of SEQ ID NO: 61; The second BCL2 of sequence number 62; BCL2 competing polynucleotide of sequence number 88; The first ESR1 primer of Sequence ID No. 65; The second ESR1 of sequence number 66; ESR1 competitive polynucleotide of sequence number 89; The first GSTM1 primer of Sequence ID No. 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 90; The first PGR primer of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of sequence number 91; The first SCUBE2 primer of Sequence ID No. 77; SCUBE2 of sequence number 78; SCUBE2 competitive polynucleotide of SEQ ID NO: 92; and The amplification of the proliferation module of the universal estrogen module probe oligonucleotide of Sequence ID No. 93, labeled with a fluorescent label for the estrogen module; The method according to any one of claims 1 to 12, wherein each module is optionally amplified by a reaction separate from the amplification of the other modules, thereby generating module-specific endpoint fluorescence.

15. During the amplification reaction, The first ACTB primer of Sequence ID No. 1 has a concentration of 33 nM or 36 nM; The second ACTB primer of Sequence ID No. 2 has a concentration of 33 nM or 36 nM; The ACTB competitive polynucleotide of SEQ ID NO: 3 is present at a concentration of 1.8e+02 copies / μL; The ACTB competitor of Sequence ID No. 101 has a concentration of 3.3e+00 copies / μL; The first GADPH primer of SEQ ID NO: 4 has a concentration of 14 nM or 106 nM; The second GADPH primer of Sequence ID No. 5 is at a concentration of 14 nM or 106 nM; The GADPH competitive polynucleotide of sequence number 6 is at a concentration of 1e+02 copies / μL; The GADPH competitive polynucleotide of sequence number 103 is at a concentration of 2.5e+00 copies / μL; The first GUSB primer of Sequence ID No. 7 has a concentration of 22 nM or 34 nM; The second GUSB primer of Sequence ID No. 8 has a concentration of 22 nM or 24 nM; The GUS competitive polynucleotide of SEQ ID NO: 9 is present at a concentration of 1.3e+02 copies / μL; The GUS competitive polynucleotide of sequence number 104 is at a concentration of 5.2e+00 copies / μL; The first RPLP0 primer of Sequence ID No. 10 has a concentration of 66 nM or 36 nM; The second RPLP0 primer of Sequence ID No. 11 has a concentration of 66 nM or 36 nM; The RPLP0 competitive polynucleotide in SEQ ID NO: 12 has a concentration of 2.9 copies / μL; the RPLP0 competitive polynucleotide in SEQ ID NO: 105 has a concentration of 1.5e+01 copies / μL; The first TRFC primer of Sequence ID No. 13 has a concentration of 84 nM or 50 nM; The second TRFC primer of Sequence ID No. 14 is at a concentration of 84 nM or 50 nM; The TRFC competitive polynucleotide of SEQ ID NO: 15 is present at a concentration of 1.4 copies / μL; The TRFC competitive polynucleotide of SEQ ID NO: 106 is present at a concentration of 1.4e+01 copies / μL; The universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label, has a concentration of 262 nM; The universal housekeeping module probe oligonucleotide of Sequence ID No. 102, labeled with a housekeeping fluorescent label, has a concentration of 314 nM; The first CD68 primer of Sequence ID No. 17 has a concentration of 23 nM or 61 nM; The second CD68 primer of Sequence ID No. 18 has a concentration of 23 nM or 61 nM; The CD68 competitive polynucleotide of SEQ ID NO: 20 is at a concentration of 0.6 copies / μL, or the CD68 competitive polynucleotide of SEQ ID NO: 110 is at a concentration of 1.6e+01 copies / μL; The CD68 probe oligonucleotide of Sequence ID No. 19, labeled with a fluorescent label for infiltration modules, is at a concentration of 28 nM; The first CTSV primer of Sequence ID No. 21 has a concentration of 28 nM or 52 nM; The second CTSV primer of Sequence ID No. 22 is at a concentration of 28 nM or 52 nM; The CTSV-competitive polynucleotide of SEQ ID NO: 24 is at a concentration of 0.013 copies / μL, or the CTSV-competitive polynucleotide of SEQ ID NO: 107 is at a concentration of 8.6e-01 copies / μL; The CTSV probe oligonucleotide of Sequence ID No. 23, labeled with a fluorescent label for infiltration modules, is at a concentration of 34 nM; The first MMP11 primer of Sequence ID No. 25 is at a concentration of 20 nM or 82 nM; The second MMP11 primer of Sequence ID No. 26 is available at concentrations of 20 nM or 82 nM; The MMP11 competitive polynucleotide of SEQ ID NO: 28 has a concentration of 2.6 copies / μL, or the MMP11 competitive polynucleotide of SEQ ID NO: 109 has a concentration of 1.3e+00 copies / μL; The MMP11 probe oligonucleotide of Sequence ID No. 27, labeled with a fluorescent label for infiltration modules, is at a concentration of 24 nM; CD68 competitive polynucleotide of SEQ ID NO: 81, concentration of 0.6 copies / μL; The CTSV-competitive polynucleotide of SEQ ID NO: 82 is present at a concentration of 0.013 copies / μL; The MMP11 competitive polynucleotide of SEQ ID NO: 83 is present at a concentration of 2.6 copies / μL; The universal invasion module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for invasion modules, has a concentration of 86 nM; The universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules, has a concentration of 234 nM; The first AURKA primer of SEQ ID NO: 29, at a concentration of 17 nM or 26 nM; The second AURKA primer of SEQ ID NO: 30 is at a concentration of 17 nM or 26 nM; The AURKA competitive polynucleotide in SEQ ID NO: 32 is at a concentration of 1.5 copies / μL; the AURKA competitive polynucleotide in SEQ ID NO: 111 is at a concentration of 6.3e-01 copies / μL; The AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for the proliferation module, is at a concentration of 20 nM; The first BIRC5 primer of Sequence ID No. 33 is at a concentration of 10 nM or 28 nM; The second BIRC5 primer of Sequence ID No. 34 is available at a concentration of 10 nM or 28 nM; The BIRC5 competitive polynucleotide in SEQ ID NO: 36 has a concentration of 0.13 copies / μL; the BIRC5 competitive polynucleotide in SEQ ID NO: 113 has a concentration of 2.9e+00 copies / μL; The BIRC5 probe oligonucleotide of SEQ ID NO: 35, labeled with a fluorescent label for the proliferation module, is at a concentration of 12 nM; The first CCNB1 primer of Sequence ID No. 37 is at a concentration of 10 nM or 27 nM; The second CCNB1 primer of Sequence ID No. 38 is at a concentration of 10 nM or 27 nM; The CCNB1 competitive polynucleotide in SEQ ID NO: 40 is at a concentration of 0.19 copies / μL; the CCNB1 competitive polynucleotide in SEQ ID NO: 116 is at a concentration of 1.0e+01 copies / μL; The CCNB1 probe oligonucleotide of Sequence ID No. 39, labeled with a fluorescent label for the proliferation module, is at a concentration of 12 nM; The concentration of the first MK167 primer in Sequence ID No. 41 is 15 nM or 44 nM; The second MK167 in Sequence ID No. 42 is at a concentration of 15 nM or 44 nM; The MK167 competitive polynucleotide of sequence number 44 is present at a concentration of 0.021 copies / μL; The MK167 competitive polynucleotide of sequence number 114 is present at a concentration of 7.4e-01 copies / μL; The MK167 probe oligonucleotide of Sequence ID No. 43, labeled with a fluorescent label for the proliferation module, is at a concentration of 19 nM; The first MYBL2 primer of Sequence ID No. 45 is at a concentration of 8 nM or 32 nM; The second MYBL2 in Sequence ID No. 46 has a concentration of 8 nM or 32 nM; The MYBL2 competitive polynucleotide of sequence number 48 has a concentration of 2.6e+03 copies / μL; The MYBL2 competitive polynucleotide of sequence number 115 has a concentration of 4.4e+00 copies / μL; The MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for the proliferation module, is at a concentration of 10 nM; The proliferation module probe oligonucleotide of Sequence ID No. 85, labeled with a fluorescent label for proliferation modules, is at a concentration of 73 nM; The proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules, has a concentration of 188 nM; The first ERBB2 primer of Sequence ID No. 49 is at a concentration of 15 nM or 20 nM; The second ERBB2 in Sequence ID No. 50 is at a concentration of 15 nM or 20 nM; The ERBB2 competitive polynucleotide of SEQ ID NO: 52 is at a concentration of 0.69 copies / μL, or the ERBB2 competitive polynucleotide of SEQ ID NO: 117 is at a concentration of 5.3e+00 copies / μL; The ERBB2 probe oligonucleotide of SEQ ID NO: 51, labeled with a fluorescent label for the HER2 module, is at a concentration of 18 nM; The first GRB7 primer of Sequence ID No. 53 is at a concentration of 54 nM or 175 nM; The second GRB7 in Sequence ID No. 54 is at a concentration of 54 nM or 175 nM; The GRB7 competitive polynucleotide of SEQ ID NO: 56 is at a concentration of 40 copies / μL, or the GRB7 competitive polynucleotide of SEQ ID NO: 119 is at a concentration of 3.6e-02 copies / μL; The GRB7 probe oligonucleotide of SEQ ID NO: 55, labeled with a fluorescent label for the HER2 module, is at a concentration of 65 nM; The universal HER2 module probe oligonucleotide of Sequence ID No. 86, labeled with a fluorescent label for the HER2 module, has a concentration of 83 nM; The universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for the HER2 module, has a concentration of 234 nM; The first BAG1 primer of Sequence ID No. 57 is at a concentration of 13 nM or 26 nM; The second BAG1 in Sequence ID No. 58 has a concentration of 13 nM or 26 nM; The BAG1 competitive polynucleotide of SEQ ID NO: 60 has a concentration of 0.039 copies / μL, or the BAG1 competitive polynucleotide of SEQ ID NO: 100 has a concentration of 1.7e+01 copies / μL; The BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for the estrogen module, is at a concentration of 16 nM; The first BCL2 primer of SEQ ID NO: 61 has a concentration of 33 nM or 34 nM; The second BCL2 of SEQ ID NO: 62, with a concentration of 33 nM or 34 nM; The BCL2 competitive polynucleotide of SEQ ID NO: 64 has a concentration of 1.9 copies / μL, or the BCL2 competitive polynucleotide of SEQ ID NO: 97 has a concentration of 1.8e+00 copies / μL; The BCL2 probe oligonucleotide of SEQ ID NO: 63, labeled with a fluorescent label for the estrogen module, has a concentration of 39 nM; The first ESR1 primer of Sequence ID No. 65 is at a concentration of 40 nM or 43 nM; The second ESR1 in Sequence ID No. 66 is at a concentration of 40 nM or 43 nM; The ESR1 competitive polynucleotide of SEQ ID NO: 68 is at a concentration of 0.83 copies / μL, or the ESR1 competitive polynucleotide of SEQ ID NO: 94 is at a concentration of 4.1e+01 copies / μL; The ESR1 probe oligonucleotide of Sequence ID No. 67, labeled with a fluorescent label for the estrogen module, is at a concentration of 48 nM; The first GSTM1 primer of Sequence ID No. 69 is at a concentration of 11 nM or 52 nM; The second GSTM1 in Sequence ID No. 70 is at a concentration of 11 nM or 52 nM; The GSTM1 competitive polynucleotide of SEQ ID NO: 72 is at a concentration of 0.075 copies / μL, or the GSTM1 competitive polynucleotide of SEQ ID NO: 99 is at a concentration of 1.9e+00 copies / μL; The GSTM1 probe oligonucleotide of SEQ ID NO: 71, labeled with a fluorescent label for estrogen modules, is at a concentration of 13 nM; The first PGR primer of Sequence ID No. 73 has a concentration of 41 nM or 57 nM; The second PGR primer of Sequence ID No. 74 has a concentration of 41 nM or 57 nM; The PGR-competitive polynucleotide of SEQ ID NO: 76 is at a concentration of 0.015 copies / μL, or the CTSV-competitive polynucleotide of SEQ ID NO: 96 is at a concentration of 4.4e+00 copies / μL; The PGR1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for the estrogen module, is at a concentration of 50 nM; The first SCUBE2 primer of Sequence ID No. 77 has a concentration of 22 nM or 54 nM; The second SCUBE2 primer of Sequence ID No. 78 is available at concentrations of 22 nM or 54 nM; The SCUBE2 competitive polynucleotide of SEQ ID NO: 80 is at a concentration of 94 copies / μL, or the SCUBE2 competitive polynucleotide of SEQ ID NO: 98 is at a concentration of 1.1e+01 copies / μL; The SCUBE2 probe oligonucleotide of SEQ ID NO: 79, labeled with a fluorescent label for the estrogen module, is at a concentration of 26 nM; The BAG1 competitive polynucleotide of sequence number 87 is at a concentration of 0.039 copies / μL; The BCL2 competitive polynucleotide of SEQ ID NO: 88 is present at a concentration of 1.9 copies / μL; The ESR1 competitive polynucleotide of SEQ ID NO: 89 is present at a concentration of 0.83 copies / μL; The GSTM1 competitive polynucleotide of SEQ ID NO: 90 is present at a concentration of 0.075 copies / μL; The PGR-competitive polynucleotide of SEQ ID NO: 91 is present at a concentration of 0.015 copies / μL; The SCUBE2 competitive polynucleotide of SEQ ID NO: 92 is at a concentration of 94 copies / μL; and / or The universal estrogen module probe oligonucleotide of Sequence ID No. 93, labeled with a fluorescent label for estrogen modules, has a concentration of 192 nM; The method according to any one of claims 1 to 14, wherein the universal estrogen module probe oligonucleotide of SEQ ID NO: 95, labeled with a fluorescent label for estrogen modules, is at a concentration of 319 nM.

16. The aforementioned subject is, HR+ HER2- Breast Cancer; Having or having previously had invasive breast cancer of anatomical stage I, II, or IIIa; and / or The method according to any one of claims 1 to 15, wherein the axillary lymph nodes numbering 0, 1, 2, or 3 or less are positive for cancer cells.

17. The fluorescent label for the housekeeping module is VIC; The fluorescent label for the growth module is FAM; The fluorescent label for the infiltration module is ABY; The fluorescent label for the HER2 module is JUN; and The method according to any one of claims 1 to 16, wherein the fluorescent label for the estrogen module is VIC.

18. The method according to any one of claims 13 to 17, further comprising the step of generating a risk score based on the endpoint fluorescence obtained after a specific number of cycles of the fluorescent labels associated with the housekeeping module, the invasion module, the growth module, the HER2 module, and the estrogen module.

19. The method according to claim 18, wherein the endpoint fluorescence is determined after 50 amplification cycles.

20. The endpoint fluorescence is determined after 50 amplification cycles. The fluorescent label for the housekeeping module is VIC; The fluorescent label for the growth module is FAM; The fluorescent label for the infiltration module is ABY; The fluorescent label for the HER2 module is JUN; and The method according to any one of claims 1 to 19, wherein the fluorescent label for the estrogen module is VIC.

21. The aforementioned risk score is calculated as follows: RSuZ=max(b_FAM ＊ (HK-FAM),-c_FAM)-b_VIC ＊ (HK-VIC)+b_ABY ＊ (HK-ABY) + max (β_JUN) ＊ (HK-JUN),-__JUN) RSu=RSuZ ＊ 1.472.99 RS=min(max(20 ＊ The method according to any one of claims 17 to 20, as determined by (RSu-6.7),0),100).

22. The method according to claim 21, wherein β_FAM = 0.35, β_VIC = 0.42, β_ABY = 0.17, β_JUN = 0.46, γ_FAM = 1.14, γ_JUN = 0.76, HK, FAM, VIC, ABY, and JUN are standardized endpoint fluorescence intensities from the associated module.

23. The fluorescent label for the housekeeping module provides a housekeeping module signal; The fluorescent label for the growth module provides a signal to the growth module; The fluorescent label for the infiltration module provides an infiltration module signal; The fluorescent label for the HER2 module provides the HER2 module signal; and The method according to any one of claims 1 to 16, wherein the fluorescent label for the estrogen module provides an estrogen module signal.

24. The aforementioned risk score is calculated as follows: Table 1 (In the formula, Prol refers to the endpoint signal from the growth module for a given sample divided by the signal from its respective control reaction; Estpl refers to the endpoint signal from the estrogen module for a given sample divided by the signal from its respective control reaction; Invp refers to the endpoint signal from the wetting module for a given sample divided by the signal from its respective control reaction; Her2 refers to the endpoint signal from the Her2 module for a given sample divided by the signal from its respective control reaction; and The method according to claim 23, wherein Hskp is determined by (referring to the endpoint signal from the housekeeping module for a given sample divided by the signal from its respective control reaction).

25. The method according to claim 24, wherein the numerical values ​​are as follows. Table 2

26. The method according to any one of claims 12 to 25, further comprising the step of determining an appropriate treatment strategy based on the risk score.

27. The method according to any one of claims 12 to 26, further comprising the step of administering one or more therapeutic agents to treat breast cancer or prevent recurrence of breast cancer, wherein the one or more therapeutic agents or steps are optionally selected from chemotherapy, radiotherapy, immunotherapy and surgery.

28. A method for determining the postoperative risk of breast cancer recurrence and / or predicting the response to chemotherapy, comprising a method for calculating a risk score according to any one of claims 12 to 25.

29. The method according to claim 28, further comprising the step of determining an appropriate treatment strategy based on the risk score.

30. The method according to either claim 28 or 29, further comprising the step of administering one or more therapeutic agents to treat breast cancer or prevent the recurrence of breast cancer.

31. A kit containing at least two polynucleotides having one of the sequences 1 to 119.

32. below: a) i) The first ACTB primer of Sequence ID No. 1; The second ACTB primer of Sequence ID No. 2; ACTB competing polynucleotide of Sequence ID No. 3; The first GADPH primer of Sequence ID No. 4; The second GADPH primer of Sequence ID No. 5; GADPH competing polynucleotide of sequence number 6; The first GUSB primer of Sequence ID No. 7; The second GUSB primer of Sequence ID No. 8; GUS competitive polynucleotide of sequence number 9; The first RPLP0 primer of Sequence ID No. 10; The second RPLP0 primer of Sequence ID No. 11; RPLP0 competitive polynucleotide of sequence number 12; The first TRFC primer of Sequence ID No. 13; The second TRFC primer of Sequence ID No. 14; TRFC competitive polynucleotide of SEQ ID NO: 15; and Universal housekeeping module probe oligonucleotide of Sequence ID No. 16, labeled with a housekeeping fluorescent label; or ii) The first ACTB primer of Sequence ID No. 1; The second ACTB primer of Sequence ID No. 2; ACTB competing polynucleotide of sequence number 101; The first GADPH primer of Sequence ID No. 4; The second GADPH primer of Sequence ID No. 5; GADPH competing polynucleotide of sequence number 103; The first GUSB primer of Sequence ID No. 7; The second GUSB primer of Sequence ID No. 8; GUS competitive polynucleotide of sequence number 104; The first RPLP0 primer of Sequence ID No. 10; The second RPLP0 primer of Sequence ID No. 11; RPLP0 competitive polynucleotide of sequence number 105; The first TRFC primer of Sequence ID No. 13; The second TRFC primer of Sequence ID No. 14; TRFC competitive polynucleotide of SEQ ID NO: 106; and Universal housekeeping module probe oligonucleotide of Sequence ID No. 102, labeled with a housekeeping fluorescent label; b) i) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 20; and CD68 probe oligonucleotide of Sequence ID No. 19, labeled with a fluorescent label for infiltration modules; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competitive polynucleotide of SEQ ID NO: 24; and CTSV probe oligonucleotide of Sequence ID No. 23, labeled with a fluorescent label for infiltration modules; and The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of SEQ ID NO: 28; and MMP11 probe oligonucleotide of sequence number 27, labeled with a fluorescent label for infiltration modules; or ii) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 81; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV competing polynucleotide of sequence number 82; The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of sequence number 83; and Universal invasion module probe oligonucleotide of Sequence ID No. 84, labeled with a fluorescent label for invasion modules; or iii) The first CD68 primer of Sequence ID No. 17; The second CD68 primer of Sequence ID No. 18; CD68 competitive polynucleotide of sequence number 108; The first CTSV primer of Sequence ID No. 21; The second CTSV primer of Sequence ID No. 22; CTSV-competitive polynucleotide of sequence number 107; The first MMP11 primer of Sequence ID No. 25; The second MMP11 primer of Sequence ID No. 26; MMP11 competitive polynucleotide of sequence number 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; c) i) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 32; and AURKA probe oligonucleotide of SEQ ID NO: 31, labeled with a fluorescent label for a growth module; The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 36; and BIRC5 probe oligonucleotide of SEQ ID NO: 35, labeled with a fluorescent label for a growth module; The first CCNB1 primer of Sequence ID No. 37; The second CCNB1 primer of Sequence ID No. 38; CCNB1 competitive polynucleotide of sequence number 40; and CCNB1 probe oligonucleotide of Sequence ID No. 39, labeled with a fluorescent label for a growth module; The first MK167 primer of Sequence ID No. 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of SEQ ID NO: 44; and MK167 probe oligonucleotide of sequence number 43, labeled with a fluorescent label for a growth module; The first MYBL2 primer of Sequence ID No. 45; MYBL2, the second element of sequence number 46; The MYBL2 competitive polynucleotide of sequence number 48; and It comprises or consists of the MYBL2 probe oligonucleotide of SEQ ID NO: 47, labeled with a fluorescent label for proliferation modules; or ii) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 32; and The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 36; and The first CCNB1 primer of Sequence ID No. 37; The second CCNB1 primer of Sequence ID No. 38; CCNB1 competitive polynucleotide of sequence number 40; and The first MK167 primer of Sequence ID No. 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of sequence number 44; and The first MYBL2 primer of Sequence ID No. 45; MYBL2, the second element of sequence number 46; The MYBL2 competitive polynucleotide of sequence number 48; and The present invention comprises or consists of a universal proliferation module probe oligonucleotide of SEQ ID NO: 85, labeled with a fluorescent label for proliferation modules. or iii) The first AURKA primer of Sequence ID No. 29; The second AURKA primer of Sequence ID No. 30; AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of Sequence ID No. 33; The second BIRC5 primer of Sequence ID No. 34; BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of Sequence ID No. 37; The second CCNB1 primer of Sequence ID No. 38; CCNB1 competitive polynucleotide of sequence number 116; and The first MK167 primer of Sequence ID No. 41; The second MK167 of sequence number 42; MK167 competitive polynucleotide of SEQ ID NO: 114; and The first MYBL2 primer of Sequence ID No. 45; MYBL2, the second element of sequence number 46; The MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; d) i) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; The ERBB2 competitive polynucleotide of SEQ ID NO: 52; and ERBB2 probe oligonucleotide of SEQ ID NO: 51, labeled with a fluorescent label for HER2 modules; The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of SEQ ID NO: 56; and GRB7 probe oligonucleotide of sequence number 55, labeled with a fluorescent label for the HER2 module; or ii) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of sequence number 52; and The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of SEQ ID NO: 56; and Universal HER2 module probe oligonucleotide of sequence number 86, labeled with a fluorescent label for HER2 modules; or iii) The first ERBB2 primer of Sequence ID No. 49; The second ERBB2 of sequence number 50; ERBB2 competitive polynucleotide of sequence number 117; and The first GRB7 primer of Sequence ID No. 53; The second GRB7 of sequence number 54; GRB7 competitive polynucleotide of SEQ ID NO: 119; and Universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; And / or e) i) The first BAG1 primer of Sequence ID No. 57; BAG1 of sequence number 58; BAG1 competitive polynucleotide of SEQ ID NO: 60; and BAG1 probe oligonucleotide of SEQ ID NO: 59, labeled with a fluorescent label for estrogen modules; The first BCL2 primer of SEQ ID NO: 61; The second BCL2 of sequence number 62; BCL2 competitive polynucleotide of SEQ ID NO: 64; and BCL2 probe oligonucleotide of SEQ ID NO: 63, labeled with a fluorescent label for estrogen modules; The first ESR1 primer of Sequence ID No. 65; The second ESR1 of sequence number 66; ESR1 competitive polynucleotide of SEQ ID NO: 68; and ESR1 probe oligonucleotide of SEQ ID NO: 67, labeled with a fluorescent label for estrogen modules; The first GSTM1 primer of Sequence ID No. 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of SEQ ID NO: 72; and GSTM1 probe oligonucleotide of SEQ ID NO: 71, labeled with a fluorescent label for estrogen modules; The first PGR primer of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of SEQ ID NO: 76; and PGR1 probe oligonucleotide of SEQ ID NO: 75, labeled with a fluorescent label for estrogen modules; The first SCUBE2 primer of Sequence ID No. 77; SCUBE2 of sequence number 78; SCUBE2 competitive polynucleotide of SEQ ID NO: 80; and SCUBE2 probe oligonucleotide of SEQ ID NO: 79, labeled with a fluorescent label for estrogen modules; or ii) The first BAG1 primer of Sequence ID No. 57; BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 87; The first BCL2 primer of SEQ ID NO: 61; The second BCL2 of sequence number 62; BCL2 competing polynucleotide of sequence number 88; The first ESR1 primer of Sequence ID No. 65; The second ESR1 of sequence number 66; ESR1 competitive polynucleotide of sequence number 89; The first GSTM1 primer of Sequence ID No. 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 90; The first PGR primer of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of sequence number 91; The first SCUBE2 primer of Sequence ID No. 77; SCUBE2 of sequence number 78; SCUBE2 competitive polynucleotide of SEQ ID NO: 92; and A universal estrogen module probe oligonucleotide of Sequence ID No. 93, labeled with a fluorescent label for estrogen modules; or iii) The first BAG1 primer of Sequence ID No. 57; BAG1 of sequence number 58; BAG1 competitive polynucleotide of sequence number 100; The first BCL2 primer of SEQ ID NO: 61; The second BCL2 of sequence number 62; BCL2 competing polynucleotide of sequence number 97; The first ESR1 primer of Sequence ID No. 65; The second ESR1 of sequence number 66; ESR1 competitive polynucleotide of sequence number 94; The first GSTM1 primer of Sequence ID No. 69; The second GSTM1 of sequence number 70; GSTM1 competitive polynucleotide of sequence number 99; The first PGR primer of Sequence ID No. 73; The second PGR of sequence number 74; PGR competitive polynucleotide of sequence number 96; The first SCUBE2 primer of Sequence ID No. 77; SCUBE2 of sequence number 78; SCUBE2 competing polynucleotide of sequence number 98; and A kit containing one or more polynucleotides of the Universal Estrogen Module Probe Oligonucleotide of Sequence ID No. 95, labeled with a fluorescent label for estrogen modules.

33. below: The first ACTB primer of SEQ ID NO: 1; the second ACTB primer of SEQ ID NO: 2; the ACTB competing polynucleotide of SEQ ID NO: 101; The first GADPH primer of SEQ ID NO: 4; the second GADPH primer of SEQ ID NO: 5; the GADPH competing polynucleotide of SEQ ID NO: 103; The first GUSB primer of SEQ ID NO: 7; the second GUSB primer of SEQ ID NO: 8; the GUSB competitive polynucleotide of SEQ ID NO: 104; The first RPLP0 primer of SEQ ID NO: 10; the second RPLP0 primer of SEQ ID NO: 11; the RPLP0 competitive polynucleotide of SEQ ID NO: 105; The first TRFC primer of SEQ ID NO: 13; the second TRFC primer of SEQ ID NO: 14; the TRFC competing polynucleotide of SEQ ID NO: 106; Universal housekeeping module probe oligonucleotide of sequence number 102, labeled with housekeeping fluorescent label. The first CD68 primer of SEQ ID NO: 17; the second CD68 primer of SEQ ID NO: 18; the CD68 competitive polynucleotide of SEQ ID NO: 110; The first CTSV primer of SEQ ID NO: 21; the second CTSV primer of SEQ ID NO: 22; the CTSV competitive polynucleotide of SEQ ID NO: 107; The first MMP11 primer of SEQ ID NO: 25; the second MMP11 primer of SEQ ID NO: 26; the MMP11 competitive polynucleotide of SEQ ID NO: 109; and Universal invasion module probe oligonucleotide of Sequence ID No. 108, labeled with a fluorescent label for invasion modules; The first AURKA primer of SEQ ID NO: 29; the second AURKA primer of SEQ ID NO: 30; the AURKA competitive polynucleotide of SEQ ID NO: 111; and The first BIRC5 primer of SEQ ID NO: 33; the second BIRC5 primer of SEQ ID NO: 34; the BIRC5 competitive polynucleotide of SEQ ID NO: 113; and The first CCNB1 primer of SEQ ID NO: 37; the second CCNB1 primer of SEQ ID NO: 38; the CCNB1 competitive polynucleotide of SEQ ID NO: 116; and The first MK167 primer of SEQ ID NO: 41; the second MK167 of SEQ ID NO: 42; the MK167 competitive polynucleotide of SEQ ID NO: 114; and The first MYBL2 primer of SEQ ID NO: 45; the second MYBL2 of SEQ ID NO: 46; The MYBL2 competitive polynucleotide of sequence number 115; and Universal proliferation module probe oligonucleotide of Sequence ID No. 112, labeled with a fluorescent label for proliferation modules; The first ERBB2 primer of SEQ ID NO: 49; the second ERBB2 of SEQ ID NO: 50; the ERBB2 competitive polynucleotide of SEQ ID NO: 117; and The first GRB7 primer of SEQ ID NO: 53; the second GRB7 of SEQ ID NO: 54; the GRB7 competitive polynucleotide of SEQ ID NO: 119; and Universal HER2 module probe oligonucleotide of sequence number 118, labeled with a fluorescent label for HER2 modules; The first BAG1 primer of SEQ ID NO: 57; the second BAG1 of SEQ ID NO: 58; the BAG1 competitive polynucleotide of SEQ ID NO: 100; The first BCL2 primer of SEQ ID NO: 61; the second BCL2 of SEQ ID NO: 62; the BCL2 competitive polynucleotide of SEQ ID NO: 97; The first ESR1 primer of SEQ ID NO: 65; the second ESR1 of SEQ ID NO: 66; the ESR1 competitive polynucleotide of SEQ ID NO: 94; The first GSTM1 primer of SEQ ID NO: 69; the second GSTM1 of SEQ ID NO: 70; the GSTM1 competitive polynucleotide of SEQ ID NO: 99; The first PGR primer of SEQ ID NO: 73; the second PGR of SEQ ID NO: 74; the PGR competing polynucleotide of SEQ ID NO: 96; The first SCUBE2 primer of SEQ ID NO: 77; the second SCUBE2 of SEQ ID NO: 78; the SCUBE2 competitive polynucleotide of SEQ ID NO: 98; and A kit containing all of the Universal Estrogen Module Probe Oligonucleotides.

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