KR102317202B1 - Method for Predicting Recurrence of Breast Cancer - Google Patents

Abstract

Predicting (or diagnosing) or predicting (or diagnosing) breast cancer prognosis (eg, recurrence or not) through analysis of protein-protein interactions between proteins related to breast cancer and/or measurement of the expression level of the protein related to breast cancer. It relates to methods and kits for doing so. In addition, the present invention relates to a method for predicting (or diagnosing) breast cancer prognosis or recurrence through HER2 protein expression level, or selecting a patient suitable for a HER2 target treatment, or a method for providing information to the prediction (or diagnosis) or selection.

Description

Method for Predicting Recurrence of Breast Cancer

Predicting (or diagnosing) or predicting (or diagnosing) breast cancer prognosis (eg, recurrence or not) through analysis of protein-protein interactions between proteins related to breast cancer and/or measurement of the expression level of the protein related to breast cancer. It relates to methods and kits for doing so. In addition, the present invention relates to a method for predicting (or diagnosing) breast cancer prognosis or recurrence through HER2 protein expression level, or selecting a patient suitable for a HER2 target treatment, or a method for providing information to the prediction (or diagnosis) or selection.

Until recently, customized diagnosis and treatment according to an individual of a disease has been mainly focused on genomic profiling. However, the cause of certain diseases, including cancer, is due to abnormal activity of cells constituting the body, more specifically, abnormal expression (eg, overexpression) of various proteins constituting and regulating cells and/or abnormal interactions between proteins. , there is a problem that is difficult to understand only by observation through genetic analysis. Accurate analysis of protein-protein interactions not only allows us to understand how intracellular signaling networks change, but also predicts the progression and characteristics of individual cancers, providing appropriate and useful information for treatment. expect to be able to

In the conventional diagnosis of breast cancer, the molecular subtype (HER2(+) or HER2(-)) is determined through IHC (Immunohistochemistry) for a specific protein set. Among them, Luminal A type or triple-negative breast cancer refers to a sample determined as HER2(-) through IHC scoring.

In general, in the case of HER2(+), targeted chemotherapy and chemotherapy are prescribed, and in the case of HER2(-), hormone therapy is prescribed. And, in the case of HER2(-), a high-risk group with a high probability of recurrence or metastasis will be prescribed with chemotherapy. In the existing relapse diagnosis of HER2(-) breast cancer, it is classified into a high-risk group and a low-risk group by predicting metastasis or recurrence using RT-PCR or microarray targeting a plurality of specific genes.

In the IHC scoring technique used for HER2 (+/-) diagnosis, the interpretation of the results based on the degree of staining may differ depending on the investigator, and the analysis technique is low in sophistication, so even in samples determined as HER2(-) as a result of IHC scoring, it is actually Significant levels of HER2 may be included, which may impede the proper treatment of patients.

There is a need to more precisely measure and quantify the HER2 expression level and utilize it for prognosis (relapse) diagnosis and treatment of breast cancer.

In the present specification, in the protein diagnostic method, it is intended to provide a technique for measuring the protein level more precisely than the traditional IHC scoring technique, and to more effectively classify a high-risk group with a high probability of recurrence.

An example is a method for predicting (or diagnosing) breast cancer prognosis or recurrence, or a method for providing information to predicting (or diagnosing) breast cancer prognosis or recurrence, comprising measuring the expression level of HER2 protein in a biological sample isolated from a breast cancer patient provides

More specifically, the method comprises:

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) Comparing with the mean value or mean ± standard deviation (SD))

may include.

The step of measuring the HER2 protein expression level of step (i) includes administering a HER2 binding material with a label attached to HER2 of a biological sample isolated from a breast cancer patient, and measuring a signal generated from the label in the near-field region. It can be carried out by steps. The near field may be determined according to a generated signal (fluorescence signal).

In the step of comparing step (ii), the signal value measured in step (i) is compared with a reference value (eg, a signal generated by reaction with a HER2 binding substance labeled in samples isolated from two or more breast cancer patients). comparing with the mean value or the mean value ± standard deviation (SD)).

The method includes, after step (ii), (iii-a) when the HER2 protein expression level (signal value) measured in step (i) is greater than or equal to the reference expression level (reference value), the reference expression level (reference value) ), as compared to the case where the biological sample is isolated from the patient, confirming that the prognosis of the breast cancer is poor, or that the probability of recurrence of breast cancer is high, and/or (iii-b) measured in step (i) When the HER2 protein expression level (signal value) is lower than the reference expression level (reference value), the breast cancer prognosis of the patient from which the biological sample is isolated is better compared to when the HER2 protein expression level is higher than the reference expression level (reference value), or It may further include the step of confirming that the likelihood of recurrence is low.

In another example, the method comprises, after step (iii-a), monitoring the prognosis or recurrence of breast cancer, or preventing and/or treating breast cancer recurrence (eg, preventing or treating breast cancer recurrence). for, drug administration, irradiation, and/or surgical operation, etc.) may be further included. In another embodiment, the method may further comprise, after step (iii-b), monitoring a breast cancer prognosis or recurrence. The monitoring of the prognosis or recurrence of breast cancer may include performing the above-described method for predicting prognosis or recurrence of breast cancer one or more times.

Another example provides a method of selecting a patient suitable for HER2 targeted therapy, or providing information for selecting a patient, comprising measuring the expression level of a HER2 protein in a biological sample isolated from a breast cancer patient.

More specifically, the method comprises:

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) average value) and comparing

may include.

The method includes the steps of, after step (ii), (iii) selecting a breast cancer patient from a biological sample having a HER2 protein expression level measured in step (i) or higher than a reference expression level as a patient suitable for HER2 target treatment. may further include.

In another example, the method may further comprise, after step (iii), (iv) performing HER2 targeted therapy to the breast cancer patient selected in step (iii).

Another example is

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient;

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) average value) and comparing

(iii) selecting a breast cancer patient from a biological sample having a HER2 protein expression level measured in step (i) equal to or higher than a reference expression level as a patient suitable for HER2 target treatment;

(iv) performing HER2 targeted therapy to the breast cancer patient selected in step (iii).

It provides a method for treating breast cancer comprising a.

Another example is

A kit for predicting or diagnosing breast cancer (prognosis or recurrence) comprising a HER2 protein expression level measuring unit including a substrate on which a substance that specifically binds to HER2 protein is immobilized on the surface, or a kit for selecting a patient suitable for HER2 target treatment to provide. The kit includes a labeled HER2 binding substance (eg, a fluorescently labeled anti-HER2 antibody, etc.) that specifically binds to the HER2 protein, and/or a signal generated by the binding (reaction) between the HER2 protein and the binding substance (eg, , a fluorescent signal, etc.) may further include a signal detecting means capable of detecting.

Another example is predicting breast cancer prognosis (or diagnosis), comprising measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient and measuring the level of HER2 and/or HER3 ), or a method of providing information in predicting (or diagnosing) of a breast cancer prognosis.

More specifically, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient, and

(II) measuring the expression level of HER2 and/or HER3 in the biological sample;

may include.

In one embodiment, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action), and

(II) As a result of the comparison of step (Ia), when the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) is higher than or equal to the reference protein-protein interaction level, the biological sample measuring the expression level of HER2 and/or HER3;

may include.

In another embodiment, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action),

(II) As a result of the comparison of step (Ia), when the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) is greater than the reference protein-protein interaction level, the biological sample measuring the expression level of HER2 and/or HER3 of

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing the average value of the expression level of HER2 or HER3)

may include.

In another embodiment, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action),

(II) As a result of the comparison of step (Ia), when the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) is greater than the reference protein-protein interaction level, the biological sample measuring the expression level of HER2 and/or HER3 of

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the mean value of the expression level of HER2 or HER3 of

(II-b) when the expression level of HER2 and/or HER3 measured in step (II) is higher than or equal to the reference expression level, the breast cancer prognosis of the patient from which the biological sample is isolated is poor compared to when the expression level is lower than the reference expression level For example, determining that the recurrence of breast cancer is high and/or (II-c) when the expression level of HER2 and/or HER3 measured in step (II) is lower than the reference expression level, the reference expression level or higher Comparing with the case, confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low

may include.

In another embodiment, the method comprises:

(II) measuring the expression level of HER2 and/or HER3 in a biological sample isolated from a breast cancer patient;

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the mean value of the expression level of HER2 or HER3 of

(I) As a result of the comparison of step (II-a), when the expression level of HER2 and/or HER3 measured in step (II) is equal to or greater than the reference expression level, the protein-protein between HER2 and PLC-gamma protein in the biological sample Steps to measure interactions

may include.

In another embodiment, the method comprises:

(II) measuring the expression level of HER2 and/or HER3 in a biological sample isolated from a breast cancer patient;

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the average value of the expression level of HER2 or HER3 of

(I) As a result of the comparison of step (II-a), when the expression level of HER2 and/or HER3 measured in step (II) is equal to or greater than the reference expression level, the protein-protein between HER2 and PLC-gamma protein in the biological sample measuring the interaction, and

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action)

may include.

In another embodiment, the method comprises:

(II) measuring the expression level of HER2 and/or HER3 in a biological sample isolated from a breast cancer patient;

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the average value of the expression level of HER2 or HER3 of

(I) As a result of comparison in step (II-a), when the expression level of HER2 and/or HER3 in step (II) is equal to or higher than the reference expression level, protein-protein interaction between HER2 and PLC-gamma protein in the biological sample measuring step,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action), and

(Ib) when the protein-protein interaction level between the HER2 and PLC-gamma protein measured in step (I) is greater than the reference protein-protein interaction level, compared to the reference protein-protein interaction level or less, the Confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is poor, such as a high probability of recurrence of breast cancer, and/or (Ic) a protein-protein between HER2 and PLC-gamma protein measured in step (I) When the interaction level is less than or equal to the reference protein-protein interaction level, compared to the case where the level of interaction is greater than the reference protein-protein interaction level, the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low steps to confirm

may include.

In another example, the method comprises, after step (II-b) or (Ib), monitoring a breast cancer prognosis, preventing, and/or treating breast cancer recurrence (eg, for preventing or treating breast cancer recurrence) , drug administration, irradiation, and/or surgical operation, etc.). In another embodiment, the method may further comprise, after step (II-c) or (I-c), monitoring a breast cancer prognosis. The monitoring of the breast cancer prognosis may include performing the above-described breast cancer prognosis predicting method at least once.

Another example is

A reaction part between HER2 and PLC-gamma protein, which includes a substrate on which a material specifically binding to HER2 is immobilized on the surface;

HER2 and/or HER3 expression level measurement unit, or

all of these

It provides a kit for predicting or diagnosing the prognosis (eg, recurrence or not) of breast cancer, including. The kit adds a signal detection means capable of detecting a signal (eg, fluorescent signal, etc.) caused by a reaction between the HER2 and PLC-gamma protein, and/or a signal material (eg, fluorescent substance)-labeled PLC-gamma protein can be included as

In the present specification, by using a protein interaction analysis technique, in a biodistributed sample isolated from a breast cancer patient, a protein related to breast cancer, such as HER2 and/or HER3 protein expression level and/or a protein between HER2 and PLC-gamma protein -Provide a diagnostic technology useful for establishing an appropriate treatment strategy for breast cancer by measuring protein interactions more precisely.

As used herein, "breast cancer prognosis" refers to all symptoms and/or conditions associated with the progression of breast cancer, for example, changes in breast cancer symptoms (improvement, alleviation, treatment (elimination), or exacerbation, etc.), whether breast cancer recurs, etc., and a good prognosis means that the symptoms of breast cancer are improved, alleviated, or treated (removed), and/or the probability of recurrence of breast cancer is low, and the prognosis is poor. Hamming may mean that the symptoms of breast cancer worsen or that there is a high probability of recurrence of breast cancer.

As used herein, "recurrence of breast cancer" refers to a general term that occurs again after breast cancer is cured, and breast cancer is cured (eg, surgical operation, drug administration, or breast cancer tissue by chemotherapy such as radiation) removal, disappearance, and/or reduction equivalent to disappearance), and the same type of tumor reoccurs at the same site (primary recurrence) or at a different site (metastatic recurrence). Breast cancer recurrence includes: ① direct recurrence in which tumor cells that have not been completely removed proliferate (eg, local recurrence and metastatic recurrence); and ② indirect recurrence, in which a new tumor of the same type occurs after tumor cells are completely removed. Recurrence of breast cancer is generally high in malignant tumors, but cases in benign tumors (eg, local recurrence) are not excluded. As used herein, "predicting or diagnosing breast cancer recurrence" may refer to confirming, determining, or determining in advance whether or not breast cancer recurrence or the possibility of recurrence in a patient whose breast cancer has been cured. In general, the possibility of recurrence of breast cancer may be determined based on approximately 5 years, 6 years, or 7 years after breast cancer treatment, but is not limited thereto.

As used herein, "HER2 targeted therapy" can be selected from all therapeutic means that specifically inactivate, suppress expression or reduce the level (concentration), and/or eliminate the HER2 protein, for example, For example, including administering a drug (eg, a small molecule compound, a HER2 target protein (eg, an antibody, etc.)) that targets the HER2 protein and/or gene (eg, that specifically inhibits the HER2 protein and/or gene) can do.

As used herein, "a patient suitable for HER2-targeted therapy" may refer to a patient for which the HER2-targeted therapy described above is predicted to be effective. It may mean that it is possible to obtain an anticancer effect (eg, reduction of cancer tissue, extinction, and/or reduction of recurrence rate, etc.) for breast cancer.

An example is a method for predicting (or diagnosing) breast cancer prognosis or recurrence, or a method for providing information to predicting (or diagnosing) breast cancer prognosis or recurrence, comprising measuring the expression level of HER2 protein in a biological sample isolated from a breast cancer patient provides

More specifically, the method comprises:

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) Comparing with the mean value or mean ± standard deviation (SD))

may include.

In the step (i) of measuring the HER2 protein expression level, a HER2 binding material to which a label is attached is administered (added) to a biological sample isolated from a breast cancer patient, and a signal generated from the label is measured in the near-field region. It can be carried out by the steps of

In the step of comparing step (ii), the signal value measured in step (i) is compared with a reference value (eg, a signal generated by reaction with a HER2 binding substance labeled in samples isolated from two or more breast cancer patients). comparing with the mean value or the mean value ± standard deviation (SD)).

The method includes, after step (ii), (iii-a) when the HER2 protein expression level (signal value) measured in step (i) is greater than or equal to the reference expression level (reference value), the reference expression level (reference value) ), as compared to the case where the biological sample is isolated from the patient, confirming that the prognosis of the breast cancer is poor, or that the probability of recurrence of breast cancer is high, and/or (iii-b) measured in step (i) When the HER2 protein expression level (signal value) is lower than the reference expression level (reference value), the breast cancer prognosis of the patient from which the biological sample is isolated is better compared to when the HER2 protein expression level is higher than the reference expression level (reference value), or It may further include the step of confirming that the likelihood of recurrence is low.

In another example, the method for predicting breast cancer prognosis or recurrence comprises, after step (iii-a), monitoring the prognosis or recurrence of breast cancer, or preventing and/or treating breast cancer recurrence (eg, breast cancer recurrence) For the prevention or treatment of, drug administration, radiation, and / or surgery, etc.) may further include. In another embodiment, the method may further comprise, after step (iii-b), monitoring a breast cancer prognosis or recurrence. The monitoring of the prognosis or recurrence of breast cancer may include performing the above-described method for predicting prognosis or recurrence of breast cancer one or more times.

Another example provides a method of selecting a patient suitable for HER2 targeted therapy, or providing information for selecting a patient, comprising measuring the expression level of a HER2 protein in a biological sample isolated from a breast cancer patient.

More specifically, the method comprises:

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) average value) and comparing

may include.

The method includes the steps of, after step (ii), (iii) selecting a breast cancer patient from a biological sample having a HER2 protein expression level measured in step (i) or higher than a reference expression level as a patient suitable for HER2 target treatment. may further include.

In another example, the method may further comprise, after step (iii), (iv) performing HER2 targeted therapy to the breast cancer patient selected in step (iii).

Another example is

(i) measuring the HER2 protein expression level in a biological sample isolated from a breast cancer patient;

(ii) comparing the HER2 protein expression level measured in step (i) to a reference expression level (e.g., the HER2 protein expression level in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) average value) and comparing

(iii) selecting a breast cancer patient from a biological sample having a HER2 protein expression level measured in step (i) equal to or higher than a reference expression level as a patient suitable for HER2 target treatment;

(iv) performing HER2 targeted therapy to the breast cancer patient selected in step (iii).

It provides a method for treating breast cancer comprising a.

Another example is

Provided is a kit for predicting or diagnosing breast cancer (prognosis or recurrence), including a HER2 protein expression level measuring unit including a substrate on which the HER2 protein is immobilized on the surface, or a kit for selecting a patient suitable for HER2 target treatment. The kit includes a labeled HER2 binding substance (eg, a fluorescently labeled anti-HER2 antibody, etc.) that specifically binds to the HER2 protein, and/or a signal generated by the binding (reaction) between the HER2 protein and the binding substance (eg, , a fluorescent signal, etc.) may further include a signal detecting means capable of detecting. The kit may be for use in the method proposed herein.

In the above steps (ii), (iii-a), and (iii-b), the reference expression level (reference value) is the mean value or mean ± standard deviation (SD) of the expression levels of HER2 in two or more reference breast cancer samples. it could mean The reference breast cancer sample may be a biological sample including breast cancer cells or breast cancer tissue isolated from a breast cancer patient, and may or may not include a test target sample. In one embodiment, the reference breast cancer sample may be a biological sample comprising breast cancer cells or breast cancer tissue isolated from two or more patients who have received breast cancer treatment (eg, surgical treatment), about 5 years after the treatment, about It may be a sample capable of confirming the prognosis or recurrence of breast cancer after 6 years or about 7 years have elapsed. The reference expression level can be databased and applied to the methods provided herein.

In one example, the expression level of the HER2 protein may be measured and/or quantified by a commonly used protein expression level measurement method (eg, ELISA, western blotting, Immunohistochemistry (IHC), etc.). For example, the expression level of the HER2 protein may be measured and/or quantified by a single-molecule pull down assay. The single-molecule pull down assay comprises: applying a sample containing the target protein to a substrate on which a target protein (HER2) is directly or indirectly (eg, via an antibody, etc.) immobilized thereon; treating and reacting a HER2 binding substance to which a labeling substance (eg, a fluorescent substance) is attached; and detecting (measurement) measuring the generated signal (eg, fluorescence signal) in the near-field region, and measuring the expression level of the target protein using the detection (measured) signal (eg, fluorescence signal) and / or quantifying may be further included. The near field may be determined according to a generated signal (fluorescence signal). The HER2 binding material may include (a) a labeled (eg, fluorescent material attached) or unlabeled capture protein that specifically binds to a HER2 protein (bait protein) and/or (b) a HER2 protein or the capture protein. It may include a labeled (eg, fluorescent substance attached) substance (eg, an antibody) that specifically binds (eg, an unlabeled capture protein).

In one embodiment, when the reference expression level of HER2 is measured by the method provided herein, the amount of protein expressed as the number of fluorescent spots measured in the 45 (um) * 90 (um) region is the total protein content in the sample. The value normalized to be 0.5 mg/ml may range from 400 to 450 or 410 to 450, 420 to 450, 430 to 450, 400 to 440 or 410 to 440, 420 to 440, or 430 to 440, but is limited thereto. it's not going to be

All steps performed in the method provided herein may be performed in cells isolated from a living body (eg, a human body) ( in vitro).

Similar to the measurement of the HER2 protein expression level, the HER3 protein expression level measurement may also be performed.

In addition to the measurement of the HER2 protein expression level and/or the HER3 protein expression level measurement as described above, a more accurate breast cancer prognosis (eg, breast cancer recurrence) by further measuring the protein-protein interaction between HER2 and a protein interacting therewith Prediction is possible.

Another example is predicting (or diagnosing) breast cancer prognosis, comprising measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient and measuring the level of HER2 and/or HER3 A method, or method of providing information in predicting (or diagnosing) of a breast cancer prognosis is provided.

More specifically, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient, and

(II) measuring the expression level of HER2 and/or HER3 in the biological sample;

may include. Steps (I) and (II) may be performed in any order.

In one example, the method may include steps (I) and (II) sequentially in the order of (I) to (II).

When the protein-protein interaction level between HER2 and PLC-gamma protein in the biological sample measured in step (I) is greater than the reference protein-protein interaction level (eg, noise value or background signal value in the measurement system), the It was confirmed that the breast cancer prognosis of the patient from whom the biological sample was isolated is poor (eg, the probability of breast cancer recurrence is high).

Accordingly, in the method, after measuring the protein-protein interaction of step (I), (Ia) the protein-protein interaction between HER2 and PLC-gamma protein of the biological sample measured in step (I) may further comprise comparing the level with a reference protein-protein interaction level (in this case, prior to the comparing step, the method may further comprise determining the reference protein-protein interaction level); Optionally, in addition, (Ib) when the protein-protein interaction level between the HER2 and PLC-gamma protein of the biological sample measured in step (I) is greater than the reference protein-protein interaction level, the reference protein-protein interaction The method may further include confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is poor, for example, a high probability of recurrence of breast cancer compared to the case of the lower level. In this case, the step of measuring the expression level of HER2 and/or HER3 in step (II) includes, as a result of the comparison of step (Ia), a protein between HER2 and PLC-gamma protein in the biological sample measured in step (I). - It may be characterized in that it is performed when the protein interaction level is greater than the reference protein-protein interaction level.

The reference protein-protein interaction level in steps (I) and/or (Ia) means a noise value or a background signal value in the measurement system, and the biological sample or the biological sample and PLC-gamma protein are not added. It may be a protein-protein interaction level measured in .

The expression level of HER2 and/or HER3 measured in step (II) is a reference expression level (eg, the expression level of HER2 or HER3 in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated. expression level) or higher, it was confirmed that the probability of breast cancer recurrence of the patient from which the biological sample was isolated is high.

Accordingly, in the method, after measuring the expression level of HER2 and/or HER3 in step (II), (II-a) based on the expression level of HER2 and/or HER3 measured in step (II) It may further comprise the step of comparing with the expression level (in this case, before the step of comparing, it may further comprise the step of measuring the reference expression level), optionally, in addition, (II-b) When the expression level of HER2 and/or HER3 measured in step (II) is higher than or equal to the reference expression level, the prognosis of the breast cancer of the patient from which the biological sample is isolated is poor, as compared to when the expression level is lower than the reference expression level, for example, breast cancer. and/or (II-c) when the expression level of HER2 and/or HER3 measured in step (II) is lower than the reference expression level, compared with the reference expression level or higher, The method may further include confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low. As described above, the step of measuring the expression level of HER2 and/or HER3 in step (II) is between HER2 and PLC-gamma protein of the biological sample measured in step (I), as a result of the comparison of step (Ia). It may be characterized in that it is performed on a biological sample having a protein-protein interaction level equal to or higher than a reference protein-protein interaction level.

In another example, the method may include sequentially the steps (I) and (II) in the order of (II) to (I). After measuring the expression level of HER2 and/or HER3 in step (II), (II-a) comparing the expression level of HER2 and/or HER3 measured in step (II) with a reference expression level may further comprise, and optionally, in addition to this, (II-b) when the expression level of HER2 and/or HER3 measured in step (II) is greater than or equal to the reference expression level, compared to when the expression level is lower than the reference expression level. , confirming that the breast cancer prognosis of the patient from which the biological sample was isolated is poor, such as a high probability of recurrence of breast cancer, and/or (II-c) the expression of HER2 and/or HER3 measured in step (II) When the level is lower than the reference expression level, the method may further include the step of confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low compared to the case where the level is higher than or equal to the reference expression level have. In this case, the step of measuring the protein-protein interaction of step (I) performed after step (II) is, as a result of the comparison of step (II-a), HER2 and HER2 of the biological sample measured in step (II) and / or when the expression level of HER3 is higher than or equal to the reference expression level, it may be characterized in that it is performed.

At this time, after measuring the protein-protein interaction of step (I), (Ia) based on the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) It may further comprise the step of comparing the protein-protein interaction level, optionally, in addition, (Ib) the protein-protein interaction level between the HER2 and PLC-gamma protein of the biological sample measured in step (I). When the reference protein-protein interaction level is greater than the reference protein-protein interaction level, compared to the reference protein-protein interaction level or less, the breast cancer prognosis of the patient from which the biological sample is isolated is poor, for example, to confirm that the recurrence of breast cancer is high Additional steps may be included. As described above, in the step of measuring the protein-protein interaction of step (I), as a result of comparison with step (II-a), the expression level of HER2 and/or HER3 in the biological sample measured in step (II) is It may be characterized in that it is performed on a biological sample that is greater than a reference expression level.

In the above steps (II), (II-a), (II-b), and (II-c), the reference expression level is the mean value or the mean value±standard deviation of the expression levels of HER2 or HER3 in two or more reference breast cancer samples. It may mean (SD). The reference breast cancer sample may be a biological sample including breast cancer cells or breast cancer tissue isolated from a breast cancer patient, and may or may not include a test target sample. In one embodiment, the reference breast cancer sample may be a biological sample comprising breast cancer cells or breast cancer tissue isolated from two or more patients who have received breast cancer treatment (eg, surgical treatment), and about 5 or 6 years after the treatment. It may be a sample in which the prognosis of breast cancer (eg, recurrence) can be confirmed after years have elapsed. In addition, the reference breast cancer sample may refer to two or more reference breast cancer samples in which the protein-protein interaction level between HER2 and PLC-gamma protein measured by the method described in step (I) is higher than or equal to the reference protein-protein interaction level. can The reference expression level can be databased and applied to the methods provided herein.

In another embodiment, when the breast cancer prognosis is breast cancer recurrence, the reference expression level is, with respect to the two or more reference breast cancer samples described above,

- "Sensitivity (%) = [(number of relapsed samples among samples with biomarker expression level greater than the reference expression level)/(total number of relapsed samples) when both steps (I) and (II) above were performed ]*100) + specificity (Specificity (%) = [(Number of non-recurrent samples among samples whose biomarker expression level is less than the reference expression level)/(Total number of non-recurrent samples)]*100) - the value obtained as 1" ( Youden's index) is 0.5 or more, 0.51 or more, 0.52 or more, 0.53 or more, 0.54 or more, 0.55 or more, or 0.56 or more, and/or

- When both steps (I) and (II) above are sequentially performed, the "positivity ratio ([Ratio of actual recurrence samples among samples determined to be positive (+) for recurrence/Actual recurrence among samples determined to be negative to recurrence] Sample ratio]) - Negative probability ratio ([Ratio of actual non-recurring samples among samples determined to be recurrent positive (+)/Ratio of actual non-recurring samples among samples determined to be recurrent negative (-)])" is 2.5 or higher, 2.75 or greater, 3 or greater, 3.25 or greater, 3.5 or greater, 3.75 or greater, or 4 or greater

It may have a level (expression level) that makes it possible. The numerical value may be databased and applied to the method provided herein.

In one embodiment, the reference expression level is the amount of protein measured by a conventional protein level measurement means, and in the case of HER2, the number of fluorescent spots measured in the 45 (um) * 90 (um) region when measured by immunofluorescence method The amount of protein expressed is normalized so that the total protein content in the sample is 0.5 mg/ml, 400 to 450 or 410 to 450, 420 to 450, 430 to 450, 400 to 440 or 410 to 440, 420 to 440, or It may range from 430 to 440.

In another example, the reference expression level may mean an average value or mean±standard deviation (SD) of the expression levels of HER2 or HER3 in two or more reference breast cancer samples. The reference breast cancer sample may be a biological sample including breast cancer cells or breast cancer tissue isolated from a breast cancer patient, and may or may not include a test target sample. In one embodiment, the reference breast cancer sample may be a biological sample comprising breast cancer cells or breast cancer tissue isolated from two or more patients who have received breast cancer treatment (eg, surgical treatment), about 5 years after the treatment, about It may be a sample capable of confirming the prognosis or recurrence of breast cancer after 6 years or about 7 years have elapsed. The reference expression level can be databased and applied to the methods provided herein.

As used herein, the expression level of a protein may mean the amount of protein in a sample, which can be clearly identified by a person of ordinary skill in the art to which the present invention pertains by a conventional method (eg, ELISA, Western blotting, Immunohistochemistry (IHC), etc.) can be measured and/or quantified.

In one example, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action),

(II) As a result of the comparison of step (Ia), when the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) is higher than or equal to the reference protein-protein interaction level, the biological sample measuring the expression level of HER2 and/or HER3, and

(II-a) comparing the expression level of HER2 and/or HER3 measured in step (II) with a reference expression level;

may include.

In another example, the method comprises:

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action),

(II) As a result of the comparison of step (Ia), when the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) is higher than or equal to the reference protein-protein interaction level, the biological sample measuring the expression level of HER2 and/or HER3;

(II-a) comparing the expression level of HER2 and/or HER3 measured in step (II) with a reference expression level, and

(II-b) when the expression level of HER2 and/or HER3 measured in step (II) is higher than or equal to the reference expression level, the breast cancer prognosis of the patient from which the biological sample is isolated is poor compared to when the expression level is lower than the reference expression level For example, determining that the recurrence probability of breast cancer is high, and/or (II-c) when the expression level of HER2 and/or HER3 measured in step (II) is lower than the reference expression level, the reference expression level Comparing with the above case, confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low

may include.

In another embodiment, the method comprises:

(II) measuring the expression level of HER2 and/or HER3 in a biological sample isolated from a breast cancer patient;

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the average value of the expression level of HER2 or HER3 of

(I) As a result of comparison in step (II-a), when the expression level of HER2 and/or HER3 in step (II) is equal to or higher than the reference expression level, protein-protein interaction between HER2 and PLC-gamma protein in the biological sample measuring, and

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action)

may include.

In another example, the method comprises:

(II) measuring the expression level of HER2 and/or HER3 in a biological sample isolated from a breast cancer patient;

(II-a) the expression level of HER2 and/or HER3 measured in step (II) above a reference expression level (eg, in a sample isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated) comparing with the average value of the expression level of HER2 or HER3 of

(I) As a result of comparison in step (II-a), when the expression level of HER2 and/or HER3 in step (II) is equal to or higher than the reference expression level, protein-protein interaction between HER2 and PLC-gamma protein in the biological sample measuring step,

(Ia) the protein-protein interaction level between the HER2 and PLC-gamma proteins of the biological sample measured in step (I) above the reference protein-protein interaction level (eg, protein-protein interaction in the case where the biological sample is not added) level of action), and

(Ib) when the protein-protein interaction level between the HER2 and PLC-gamma protein measured in step (I) is greater than the reference protein-protein interaction level, compared to the reference protein-protein interaction level or less, the Confirming that the breast cancer prognosis of the patient from which the biological sample is isolated is poor, such as a high probability of recurrence of breast cancer, and/or (Ic) a protein-protein between HER2 and PLC-gamma protein measured in step (I) When the interaction level is less than or equal to the reference protein-protein interaction level, compared to the case where the level of interaction is greater than the reference protein-protein interaction level, the breast cancer prognosis of the patient from which the biological sample is isolated is good, for example, the probability of recurrence of breast cancer is low steps to confirm

may include.

In another embodiment, the method comprises, after step (II-b) or (Ib), monitoring a breast cancer prognosis, preventing, and/or treating breast cancer recurrence (eg, preventing or treating breast cancer recurrence) for, drug administration, irradiation, and/or surgical operation, etc.) may be further included. In another embodiment, the method may further comprise monitoring the prognosis of breast cancer after step II-c) or (I-c). The monitoring of the breast cancer prognosis may include performing the above-described breast cancer prognosis predicting method at least once.

Another example is after (eg, after step (II-b) or (Ib)) or after the above-described method of predicting (or diagnosing) whether prognosis of breast cancer, or the method of providing information to predicting (or diagnosing) of prognosis of breast cancer. Drug administration, radiation, and/or surgical operation for the prevention or treatment of breast cancer recurrence in patients identified in (II-b) or (Ib) with a poor prognosis of breast cancer, such as a high probability of recurrence of breast cancer It provides a method for preventing and/or treating breast cancer or breast cancer recurrence, comprising performing can The breast cancer patient has or has been diagnosed with breast cancer, a patient who has received breast cancer treatment (eg, surgery, drug administration, chemotherapy such as radiation), and/or a patient whose breast cancer is cured (eg, surgical operation, drug It may be a patient who has been reduced to a degree equivalent to the removal, disappearance, and/or annihilation of breast cancer tissue by chemotherapy such as administration, radiation, etc.). The patient may be a mammal such as a human, and may be a target for predicting or diagnosing the prognosis or recurrence of breast cancer. said breast cancer is (1) HER2 negative (-) breast cancer, (2) hormone receptor (HR; eg, estrogen receptor (ER) and/or progesterone receptor (PR)) positive (+) breast cancer, or (3) said breast cancer It may be breast cancer having both the characteristics of (1) and (2). The breast cancer patient may be a patient identified (or diagnosed, or determined) as a breast cancer patient determined (confirmed) to be HER2(-), and/or determined (confirmed) to ER/PR(+) and HER2(-). have. The methods described herein are HER2 negative (-), and/or hormone receptor (HR) (eg, estrogen receptor (ER) and/or progesterone receptor (PR)) positive (+) confirmed (diagnosed, diagnosed) breast cancer It may be performed on a patient.

The determination of the positive (+) and negative (-) may be determined by a conventional protein level measurement method, for example, immunity using an ER-specific antibody, a PR-specific antibody, and/or a HER2-specific antibody It can be determined by the score (IHC score) determined by histochemical staining (IHC) (eg, when determining the IHC score (0-3), ER / PR (+) is IHC score (intensity) ≥ 2 (2 or 3), HER2(-) may be the case where IHC score(intensity)≤1 (0, or 1+)).

In addition to performing step (i), the method may further comprise measuring the expression level of (a) estrogen receptor (ER) and/or (b) PD-1 and/or CD3e in the sample. In this case, when (a) the expression level of ER in the sample is high and/or (b) the expression level of PD-1 and/or CD3e is low, compared with the case where the expression level of PD-1 and/or CD3e is not, the prognosis of breast cancer is poor or the possibility of breast cancer recurrence can be determined to be high. The evaluation (high or low) of the ER expression level and the expression level of PD-1 and/or CD3e may be evaluated in comparison with a reference expression level, wherein the reference expression level is, for example, as analyzed by IHC, ER expression level in HER2(-) cells (eg, SKBR3), or expression level of PD-1 and/or CD3e, but is not limited thereto.

In determining the ER / PR level, a conventional anti-ER antibody (eg, clone 6F11, 1:200 dilution, Novocastra, Bond-Max system) and an anti-PgR antibody (eg, clone 16, 1:800 dilution, Novocastra) , using the Bond-Max system), and scoring the stained nuclei (not cytoplasm) (usually according to IHC scoring; intensity score (0-3) and proportion score (0-5)), and the values It can be judged as positive when it is equal to or greater than 1% of cancer cells, and negative when less than 1% of cancer cells, but is not limited thereto. Also, in determining the level of HER2, immunostaining using an anti-HER2 antibody (eg, 4B5, Ventana, BenchMark XT), and scoring the stained nuclei (not cytoplasm) (generally according to IHC scoring), then was evaluated on the basis of:

Only the membrane staining intensity and pattern is evaluated using the new recommendations of the American Society of Clinical Oncology(ASCO)/College of American Pathologists (CAP) (2013 update, from 2013.11.01): Circumferential membrane staining that is complete, intense, and within　 > 10% of tumor cells results in a score of "3+". Circumferential membrane staining that is incomplete and/or weak/moderate and within > 10% of tumor cells or Complete and circumferential membrane staining that is intense, and within >= 10% of tumor cells is scored "2+". Incomplete membrane staining that is faint/barely perceptible and within >10 %　of tumor cells is "1+". No staining is observed or membrane staining that is incomplete　and is faint/barely perceptible and within >= 10% of tumor cells is "0". A positive test is defined as staining of 3+ score. The score of 2+ is　interpreted as equivocal. And a negative test is defined as staining of 0/1+ scores.

As used herein, the term "protein-protein interaction (PPI)" may refer to a physical and/or chemical bond or complex formation between a first protein and a second protein, for example, It may be measured by one or more of factors such as binding frequency, binding strength (strength), and binding time. In addition, the interaction (binding) between the first protein (HER2) and the second protein (PLC-gamma or anti-HER-2 antibody) is not only a direct interaction (binding) between them, but also other proteins (in the signaling pathway) Interactions (bindings) mediated by a protein located between the first protein and the second protein may also be included. The protein-protein interaction herein may be a single molecule reaction (reaction between one molecule of a first protein and one molecule of a second protein).

As used herein, HER2, estrogen receptor (ER), PD-1, CD3e, EGFR, HER3, and/or PLC-gamma are each independently derived from mammals such as humans, primates such as monkeys, mice, and rodents such as rats. may be derived.

For example, the HER2 is GenBank Accession No. It may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in X03363.1 and the like. EGFR is GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1, or the like. HER3 is GenBank Accession No. It may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_001982 and the like. PLC-gamma (phospholipase C (PLC)-gamma; for example, PLC-gamma 1) may be derived from mammals such as primates such as humans and monkeys, and rodents such as mice and rats, for example, GenBank Accession No. . A protein having an amino acid sequence provided in NP_002651.2, NP_877963.1, NP_037319.1, etc., or a SH2 domain thereof (Src homology 2 domain: comprising at least one of an N-terminal SH2 domain and a C-terminal SH domain; for example, NP_037319. 1, the 545th to 765th amino acid sequence region, or the 540th or 545th to 765th amino acid sequence region in NP_002651.2).

In the present specification, measurement of the expression level of HER2 protein, estrogen receptor (ER), PD-1, and/or CD3e (hereinafter, referred to as a first protein) may be performed by protein-protein interaction, for example, the first It may be measured as one or more of factors such as a frequency of physical and/or chemical bonding (interaction) between a protein and a second protein binding thereto, binding strength (strength), and binding time. In addition, the binding between the first protein and the second protein is not only a direct binding between them, but also another protein in the middle (a third protein; a protein that binds to the first protein, in this case, the second protein is a protein that binds to a third protein) may also include a linkage via the The protein-protein interaction herein may be a single molecule reaction (reaction between one molecule of a first protein and one molecule of a second protein).

In one example, the expression level of the first protein (HER2 protein) means quantifying the amount of the first protein (HER2 protein) in the sample, which is a sample on a substrate on which a material specifically binding to the first protein is immobilized. treatment to immobilize the first protein on a substrate, add a labeled detecting antibody (second protein) thereto, and measure the protein-protein interaction therebetween to quantify it. The protein-protein interaction can be quantified by measuring a signal generated from a label attached to the second protein.

The step of measuring a protein-protein interaction between a first protein (HER2) and a second protein (eg, a HER2 binding antibody) performed in the methods provided herein may be performed in an isolated cell or tissue in vitro or extracellularly ( in vitro ) may be performed. In addition, the step of measuring the protein-protein interaction between the first protein (HER2) and the second protein (PLC-gamma) performed in the method provided herein may be performed in vitro or extracellularly ( in vitro ) may be performed.

Hereinafter, in the description of the step of measuring the protein-protein interaction, the first protein refers to HER2 and the second protein refers to a HER2-binding antibody or PLC-gamma.

Measuring the protein-protein interaction between the first protein and the second protein may include the following steps:

(1) A biological sample isolated from a breast cancer patient containing a first protein is added to a substrate including a material (eg, anti-HER2 antibody) that specifically binds to the first protein on the surface to immobilize the first protein preparing a substrate;

(2) reacting by adding a labeled (labeled material) second protein to the prepared substrate on which the first protein is immobilized; and

(3) measuring a signal from the reactant obtained in step (2) (measurement of protein-protein interaction).

Optionally, the step of measuring the protein-protein interaction, in addition to the steps (1) to (3) described above,

(4) obtaining a signal value for the unit amount of the biological sample used in step (1) and/or the first protein unit amount in the biological sample using the signal measured in step (3); may include

The signal value for the unit amount of the biological sample may be obtained by dividing the signal value measured in step (3) by the amount of the biological sample used in step (1), and the signal value for the first protein unit amount is After dividing the signal value measured in step (3) by the amount of the first protein in the biological sample used in step (1) or by the amount of the biological sample used in step (1), the first protein in the sample It may be obtained by dividing the amount.

In the present specification, the protein-protein interaction level is the signal value measured in step (3) or a value obtained by dividing the signal value by the amount of the biological sample used and/or the amount of the first protein (HER2) in the biological sample can mean

Hereinafter, the above steps will be described in more detail:

Step (1): Preparing the substrate on which the first protein is immobilized

The step (1) is a step of preparing a substrate on which the first protein is immobilized by applying a biological sample including the first protein to the surface of the substrate including a material that specifically binds to the first protein.

The first protein may be HER2.

The biological sample may be a cell (eg, cancer cell), tissue (eg, cancer tissue), a lysate, lysate, or extract of the cell or tissue isolated from a breast cancer patient, a body fluid (eg, blood (whole blood, plasma, or serum)) , saliva, etc.). When the biological sample is a tissue (eg, cancer tissue), a ^{size of at least 125 mm 3} for pulverization is sufficient, and the amount of tissue sample required for one measurement is in the range of about 1/50 to about 1/75 ^{of at least 125 mm 3 or more.} can, but is not limited thereto. When the biological sample is a cell (cancer cell), the amount of the cell sample required for one measurement is about 10 cells to 10 ¹⁰ cells, about 10 cells to 10 ⁷ cells, about 10 cells to 10 ⁵ cells, about 10 ³ cells to 10 ¹⁰ cells, about 10 ³ cells to 10 ⁷ cells, about 10 ³ cells to 10 ⁵ cells, for example ^{, may be about 10 4} ± 50 cells, but is not limited thereto. It is a value that can be appropriately determined by the person. The breast cancer patient may be a mammal such as a human who has been diagnosed and/or treated with breast cancer, and may be a target for predicting or diagnosing the prognosis (eg, recurrence) of breast cancer.

The substrate may have any material and/or any structure capable of immobilizing the first protein on the surface (either crystalline or amorphous may be used). In one example, the substrate may be made of a material having a refractive index of light (about 1.3) or higher of water, which accounts for a large portion of a biomaterial, in consideration of ease of detecting a label signal. In one example, the substrate may have a thickness of about 0.1 to about 1 mm, about 0.1 to about 0.5 mm, 0.1 to about 0.25 mm, or about 0.13 to about 0.21 mm, and a refractive index of about 1.3 to about 2, about 1.3. to about 1.8, about 1.3 to about 1.6, or about 1.5 to about 1.54. The substrate may be any material that satisfies the refractive index range, for example, may be obtained from a material selected from the group consisting of glass (refractive index: about 1.52), quartz, and the like, but is not limited thereto. The substrate may have any shape commonly used for observation of biological samples, and may be, for example, a well type, a slide type, a channel type, an array type, a microfluidic chip, a microtubule (capillary), etc., but is limited thereto. it's not going to be When observing a fluorescence microscope, it can be observed by mounting a cover glass on the substrate to which the sample is applied, the material of the cover glass is as described above for the substrate, and the thickness may be in the range or thinner than that described in the substrate (eg, refractive index 1.52, may be 0.17 mm thick, but not limited thereto).

The substance specifically binding to the first protein may be selected from all substances capable of specifically binding to the first protein (HER2), for example, an antibody or antigen binding thereof that specifically binds to the first protein. One selected from the group consisting of fragments (eg, scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2 of antibodies), aptamers (protein or nucleic acid molecules), small molecule compounds, and the like may be more than In this case, the material specifically binding to the first protein has a site that does not interfere with the interaction between the first protein and the second protein, that is, a site where the first protein and the second protein interact (binding). It may be binding to the first protein at a site that is not.

In one example, the substrate is appropriately surface-modified to include (immobilize) a biological material (eg, an antibody, etc.) that specifically binds to the first protein on the surface, or specifically to the first protein directly on the surface The binding material may be fixed. When the substrate is surface-modified, the substrate may be treated (eg, coated) with any compound having a functional group capable of immobilizing a biological material (eg, an antibody, etc.) that specifically binds to the first protein on one surface. and, for example, may be treated with a compound containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group, and an amine group. In one embodiment, the compound comprising a functional group selected from the group consisting of an aldehyde group, a carboxyl group and an amine group is biotin, bovine serum albumin (BSA), bovine serum albumin to which biotin is bound, polyethylene glycol (polyethylene) It may be one or more selected from the group consisting of glycol; PEG), biotin-bound PEG (polyethylene glycol-biotin; PEG-biotin), and polysorbate (eg, Tween20), but is not limited thereto. The surface-treated substrate may be further treated (eg, coated) with at least one selected from the group consisting of neutravidin, streptavidin, avidin, and the like.

Step (2): reacting the first protein and the second protein

Step (2) is a step of reacting by adding a labeled second protein to the prepared substrate on which the first protein is immobilized.

The second protein may be a HER2 binding antibody.

The labeled second protein is labeled with a labeling agent that generates a detectable signal by the second protein (the labeling agent is bound, e.g., chemically (e.g., covalently or non-covalently), recombinantly, or physically) , it may refer to a form in which a tag to which a label can be bound is attached. The detectable signal may be selected from all signals (eg, light, radiation, etc.) that can be measured through conventional enzymatic reactions, fluorescence, luminescence, and/or radiation detection. The labeling material may be at least one selected from the group consisting of all small molecule compounds, proteins, peptides, nucleic acid molecules, and the like capable of generating the labeling signal, for example, fluorescent dyes (small molecule compounds; Cyanine, Alex, Dylight, Fluoprobes, etc.). ), fluorescent proteins (eg, green fluorescent protein (GFP, enhanced GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BPF), red fluorescent protein (RPF), etc.) It may be one or more selected from. The tag may be one or more selected from all types commonly used, such as His-tag / Ni-NTA. The concentration of the labeling substance used may be appropriately determined in the range of about 1 uM or less in order to enable accurate and easy detection without generating noise, for example, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, It may be about 10nM to 1000nM, 10nM to 500nM, or 10nM to 100nM, but is not limited thereto. The signal generated from the labeling material as described above can be measured by any signal detection means (eg, a conventional fluorescence microscope, a fluorescence camera, a fluorescence intensity measurement (quantitation) device, etc.) commonly used to detect or measure it.

In order to more accurately measure the interaction between the first protein and the second protein, between the reacting step (step (2)) and the subsequent protein-protein interaction measurement step (step (3)), the substrate on which the reaction occurred It may further comprise the step of washing in a conventional method.

Step (3): protein-protein interaction measurement step

The step (3) is a step of measuring a signal from the reactant obtained in the step (2). The signal measurement is any means capable of detecting (or measuring or confirming) the label signal (eg, a signal measurable through a conventional method such as enzymatic reaction, fluorescence, luminescence, or radiation detection) used in step (2). can be performed using

In one example, the protein-protein interaction measurement in step (3) may be by real-time analysis.

In one example, when the label signal is a fluorescence signal, the signal detection is a fluorescence signal generated by supplying a light source absorbed by the label material generating the fluorescence signal, for example, a fluorescence microscope, a fluorescence camera, and/or fluorescence. Intensity measurement (quantitation) devices and the like may be used to image and/or quantify.

In one embodiment, when the signal is a fluorescence signal, the fluorescence signal may be imaged and/or quantified using a fluorescence camera.

When the signal is a fluorescent signal, step (3) (protein-protein interaction measurement step) is

(a) supplying a light source to the reactant of step (2); and

(b) detecting a fluorescence signal generated by the supplied light source

may include.

The step of supplying the light source in step (a) is a step of supplying a light source to the reaction product of the first protein and the second protein obtained in step (2). If this purpose can be achieved, the supply timing of the light source is There are no special restrictions. For example, the light source may be continuously supplied from before, simultaneously, or after step (1) to after step (2), or may be supplied for a predetermined time immediately before, simultaneously, or immediately after step (2), but is limited thereto it is not

The light source may be any light source having a wavelength corresponding to a fluorescent signal, for example, a laser, a halogen lamp, or the like.

The wavelength of the light source may be adjusted according to the fluorescence signal used, for example, may be selected from about 300 nm to about 600 nm or from about 350 nm to about 560 nm. More specifically, green fluorescent protein absorbs about 480 nm, yellow fluorescent protein absorbs about 540 nm, blue fluorescent protein absorbs about 375 nm, 　 cyan fluorescent protein absorbs about 425 nm, so green fluorescence as a fluorescence signal When used, the wavelength of the light source is about 460 to about 500 nm, when yellow fluorescence is used as the fluorescence signal, the wavelength of the light source is about 520 to about 560 nm, and when blue fluorescence is used as the fluorescence signal, the wavelength of the light source is about 350 to about 400 nm, when cyan fluorescence is used as the fluorescence signal, the wavelength of the light source may be selected from about 400 to about 450 nm. The above-described wavelength may be the proximity light for each fluorescent material.

In one embodiment, in the protein-protein interaction measurement step of step (3), a light source is supplied using a total internal reflection fluorescence (TIRF) microscope or a confocal microscope, and a fluorescence signal is performed in a conventional manner. This can be done by observing In another example, by using the total reflection fluorescence microscope equipped with a fluorescence camera for signal imaging, such as an electron-multiplying charge-coupled device (EMCD) camera or a complementary metal oxide semiconductor (CMOS) camera, a light source is supplied and the fluorescence signal is imaged and/or quantification.

In the following, a case in which the protein-protein interaction measurement step of step (3) is performed using a total reflection microscope and a fluorescence camera will be described in more detail by way of example:

a) Mount the substrate of step (1) or step (2) on a total reflection microscope. In a total reflection microscope, the supply position of the light source is usually downward, and depending on the type of total reflection microscope, the fluorescence signal is transmitted above the substrate (in this case, from bottom to top, in the direction of the light source supply unit, the substrate, the lens, or the substrate, the light source supply unit, and the lens). may be located in that order) or from below (in this case, from bottom to top, the lens, the light source supply, the substrate, or the light source supply, the lens, the substrate, or the lens, the substrate, the light source supply may be located in this order) can be observed.

b) The light source may be a laser, and the intensity of the light source is from about 0.5 mW to about 5 mW, from about 0.5 mW to about 4.5 mW, from about 0.5 mW to about 4 mW, from about 0.5 mW to about 3.5 mW, from about 0.5 mW to about 3 mW, about 0.5 mW to about 2.5 mW, about 0.5 mW to about 2 mW, about 1 mW to about 5 mW, about 1 mW to about 4.5 mW, about 1 mW to about 4 mW, about 1 mW to about 3.5 mW , about 1 mW to about 3 mW, about 1 mW to about 2.5 mW, about 1 mW to about 2 mW, about 1.5 mW to about 5 mW, about 1.5 mW to about 4.5 mW, about 1.5 mW to about 4 mW, about 1.5 mW to about 3.5 mW; It can be appropriately selected according to the signal or the label material used, and/or the configuration of the equipment (eg, when an attenuation filter is used, a light source with high intensity may be used).

C) The fluorescence signal generated by the supply of the light source may be imaged and/or quantified by photographing it with a fluorescence camera.

The imaging (or imaging) of the fluorescence signal may be performed simultaneously with the supply of a light source or within the duration of the signal generation in consideration of the fluorescence signal generation and maintenance time (luminescence time, lifetime) of the labeling material.

In photographing (or imaging) a fluorescence signal with a fluorescence camera (eg, EMCCD camera), the exposure time per frame, laser power, camera gain value, total shooting frame, and the like can be appropriately adjusted. For example, the shorter the exposure time per frame is, the less signals accumulated in one frame. In order to offset this, the laser power or the sensitivity of the fluorescent camera may be increased. In one example, the exposure time per frame is about 0.001 second to about 5 seconds, about 0.001 second to about 3 seconds, about 0.001 second to about 2 seconds, about 0.001 second to about 1 second, about 0.001 second to about 0.5 second, about 0.001 second to about 0.3 second, about 0.001 second to about 0.1 second, about 0.01 second to about 5 second, about 0.01 second to about 3 second, about 0.01 second to about 2 second, about 0.01 second to about 1 second, about 0.01 seconds to about 0.5 seconds, about 0.01 seconds to about 0.3 seconds, about 0.01 seconds to about 0.1 seconds, about 0.05 seconds to about 5 seconds, about 0.05 seconds to about 3 seconds, about 0.05 seconds to about 2 seconds, about 0.05 seconds to about 1 second, about 0.05 second to about 0.5 second, about 0.05 second to about 0.3 second, about 0.05 second to about 0.1 second, about 0.07 second to about 5 second, about 0.07 second to about 3 second, about 0.07 second seconds to about 2 seconds, about 0.07 seconds to about 1 second, about 0.07 seconds to about 0.5 seconds, about 0.07 seconds to about 0.3 seconds, about 0.07 seconds to about 0.1 seconds, about 0.1 seconds to about 5 seconds seconds, from about 0.1 seconds to about 3 seconds, from about 0.1 seconds to about 2 seconds, from about 0.1 seconds to about 1 second, from about 0.1 seconds to about 0.5 seconds, or from about 0.1 seconds to about 0.3 seconds, such as about 0.1 seconds; However, the present invention is not limited thereto.

For example, in the case of using an EMCCD camera, photons generated from a labeling material (eg, eGFP) are converted into electrons through a device of the EMCCD and measured (photoelectric effect). At this time, the number of electrons generated per photon can be changed through the gain value. As the set gain value increases, the number of electrons per photon increases, which increases the sensitivity of the EMCCD and also increases the background noise. Therefore, the signal-to-noise ratio is important. In one embodiment, in order to obtain a good signal-to-noise ratio, the gain is set to about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 50, about 20 to about 100, about 20 to about 80, about 20 to about 60, about 20 to about 50, about 30 to about 100, about 30 to about 80, about 30 to about 60, or about 30 to about 50, such as about 40, , is not limited thereto, and may be appropriately selected in consideration of camera sensitivity, lifespan, equipment construction status, noise, test conditions, and the like.

The total number of frames is multiplied by the exposure time to get the total shooting time (exposure time * total number of frames = shooting time). Since the fluorescent signal disappears after the emission time of the fluorescent material, the total number of photographing frames and/or the exposure time may be adjusted so that the photographing time proceeds within the emission time.

In order to increase the accuracy of the protein-protein interaction measurement result, the imaging is performed at one or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, or 10 or more (the upper limit is the size of the substrate. (determined according to the size and imageable area) of the substrate or one or more channels including the same (each channel includes two or more substrates), and obtain the number of spots (also called PPI complexes) where the signal appears, or and/or quantify the obtained fluorescence signal, which can be viewed as quantifying protein-protein interaction.

In another example, the protein-protein interaction may be quantified by quantifying the fluorescence intensity measured in step (3) using a conventional apparatus.

Step (4): biological sample unit amount and/or a first protein in unit amount Measuring the level of protein-protein interaction for

Step (4) is to obtain a protein-protein interaction level for the unit amount of the biological sample added in step (1) and/or the unit amount of the first protein in the biological sample using the signal measured in step (3) may be a step.

The level of protein-protein interaction for a unit amount of a biological sample and/or a first protein refers to a step (unit weight or concentration (eg, 1 ug/ml)) of a biological sample and/or a first protein. 3) to mean the measured signal value (quantified value of signal or signal strength),

(a) dividing the signal value measured in step (3) by the amount (weight, concentration or fluorescence signal) of the biological sample and/or the first protein in the biological sample,

(b) an increase in the signal value measured in step (3) according to an increase in the amount (weight, concentration or fluorescence signal) of the biological sample and/or the biological sample (ie, the biological sample and/or the biological sample) It can be obtained by calculating the slope of the graph obtained by using the amount (weight, concentration, or fluorescence signal) of the first protein in the x-axis and the signal value measured in step (3) as the y value.

In one embodiment, in the step (4) measuring the protein-protein interaction level for the unit amount, the signal value measured in step (3) is the amount (weight, concentration, or fluorescence signal) of the biological sample used in step (1) ) may be included.

In another embodiment, the step (4) measuring the protein-protein interaction level per unit amount comprises:

- dividing the signal value measured in step (3) by the amount (weight, concentration or fluorescence signal) of the first protein in the biological sample used in step (1);

- Obtaining a signal value for a unit amount of a biological sample by dividing the signal value measured in step (3) by the amount (weight, concentration, or fluorescence signal) of the biological sample used in steps (1) to (1) (4) -1), and dividing the signal value for the unit amount of the biological sample obtained in step (4-1) by the amount (weight, concentration, or fluorescence signal) of the first protein in the biological sample, the first included in the biological sample It may include a step (4-2) of obtaining a signal value for a unit amount of protein.

The method provided herein may further include normalizing the measured value obtained in step (I) and/or (II) in order to increase the accuracy thereof.

The normalization step includes measuring the HER2 protein expression level in step (i), the protein-protein interaction level in step (I) and/or the HER2 and/or HER3 protein expression level in step (II) as a whole between samples. It may be to proceed by setting the protein amount or concentration to be the same at a predetermined value. In this case, the total protein amount or concentration for standardization in step (I) and step (II) may be the same or different from each other. In addition, the total protein amount or concentration for the standardization may be arbitrarily and appropriately determined so that it is the same between samples.

In the present specification, the expression level of a protein or the amount of protein such as HER2, HER3, EGFR, etc. may refer to the amount of protein in a biological sample, which may be measured by any conventional method, for example, a conventional immunoblotting method (such as , quantitative western blotting), ELISA (enzyme-linked immunosorbent assay; direct assay, indirect assay, sandwich assay, etc.), but is not limited thereto. In one example, a biological sample is processed on a substrate on which a material specifically binding to the protein is immobilized, the protein is immobilized on the substrate, a labeled detection antibody is added thereto, and a signal generated from the label is detected. The protein may be quantified by measuring, but is not limited thereto.

In another example, a kit for predicting or diagnosing breast cancer (prognosis or recurrence) comprising a HER2 protein expression level measuring unit including a substrate on which a substance specifically binding to HER2 protein is immobilized on the surface, or a patient suitable for HER2 target treatment A kit for screening is provided. The kit includes a labeled HER2 binding substance (eg, a fluorescently labeled anti-HER2 antibody, etc.) that specifically binds to the HER2 protein, and/or a signal generated by the binding (reaction) between the HER2 protein and the binding substance (eg, , a fluorescent signal, etc.) may further include a signal detecting means capable of detecting.

In another example, breast cancer comprising a reaction part between HER2 and PLC-gamma protein comprising a substrate on which a substance specifically binding to HER2 is immobilized on a surface, a part measuring the expression level of HER2 and/or HER3, or both A kit for predicting or diagnosing the prognosis (eg, whether recurrence) is provided. The kit adds a signal detection means capable of detecting a signal (eg, fluorescent signal, etc.) caused by a reaction between the HER2 and PLC-gamma protein, and/or a signal material (eg, fluorescent substance)-labeled PLC-gamma protein can be included as

Another example is

Provided is a kit for predicting or diagnosing breast cancer (prognosis or recurrence), including a HER2 protein expression level measuring unit including a substrate on which the HER2 protein is immobilized on the surface, or a kit for selecting a patient suitable for HER2 target treatment. The kit includes a labeled HER2 binding substance (eg, a fluorescently labeled anti-HER2 antibody, etc.) that specifically binds to the HER2 protein, and/or a signal generated by the binding (reaction) between the HER2 protein and the binding substance (eg, , a fluorescent signal, etc.) may further include a signal detecting means capable of detecting. In the kit, the HER2 protein expression level measuring unit is a part in which a signal is generated by a reaction between HER2 and a labeled HER2 binding material, and is a well (eg, multi-well) type, a slide type, a channel type, an array type, and a microfluidic type. It may be in the form of a chip, microtubule (capillary), etc., but is not limited thereto.

Another example is

A reaction part between HER2 and PLC-gamma protein, which includes a substrate on which a material specifically binding to HER2 is immobilized on the surface;

HER2 and/or HER3 expression level measurement unit, or

all of these

It provides a kit for predicting or diagnosing the prognosis (eg, recurrence or not) of breast cancer, including. The kit further includes a signal detection means capable of detecting a signal (eg, a fluorescent signal, etc.) caused by a reaction between the PLC-gamma protein and/or the HER2 and the PLC-gamma protein labeled with a label (eg, a fluorescent substance) may include The reaction unit between the HER2 and PLC-gamma protein may be a well (eg, multi-well) type, a slide type, a channel type, an array type, a microfluidic chip, a microtubule (capillary), and the like, but is not limited thereto. The substance specifically binding to the substrate and HER2 is the same as described in step (1) above, and may be, for example, an anti-HER2 antibody. The labeling material for labeling the PLC-gamma protein is the same as described above in step (2).

In the kit provided herein, the signal detecting means may be any commonly used signal detecting means depending on the signal generated by the used labeling material. For example, the signal detection means may include a signal stimulation unit and a signal detection unit, and in addition to this, may further include a signal analysis unit to analyze (eg, quantify or image) the measured signal. In one example, signal stimulation, signal detection, and signal analysis may be performed at different sites, or at least two or more of these may be performed simultaneously or sequentially at one site. In one example, when the label is a fluorescent material, the signal detecting means may be selected from all means capable of generating and detecting a fluorescent signal, for example, a fluorescent signal stimulating unit (eg, a light source), and a fluorescent signal. It may include a detection unit and/or a fluorescence signal analysis unit. In one embodiment, the signal detecting means includes, or in addition to, a total internal reflection fluorescence (TIRF) microscope or a confocal microscope (for detecting a light source and a fluorescence signal), a fluorescence camera, such as an EMCCD (Electron -Multiplying charge-coupled device) cameras or complementary metal oxide semiconductor (CMOS) cameras may be further included to supply a light source and perform imaging and/or quantification of a fluorescence signal. The wavelength, intensity, and fluorescent camera measurement conditions of the light source (eg, exposure time per frame, laser power, camera gain value, total shooting frame, etc.) are the same as described above in step (3).

The method provided herein is by measuring the expression level of a predetermined biomarker and/or measuring and examining the PPI between proteins interacting with the biomarker, thereby predicting the prognosis of a breast cancer patient, in particular, whether relapse or not, or the patient It is expected to be useful as a diagnostic technology for personalized treatment for each patient, since it enables a more effective treatment strategy to be established by selecting a treatment method suitable for each individual.

1 is a schematic diagram of a protein-protein interaction (PPI) measurement method.
2A is a graph showing HER2-PLCG1 PPI counts measured for 73 samples in one embodiment.
Figure 2b is a graph showing the EGFR-Grb2 PPI count measured for 73 samples in one embodiment.
3A is a graph showing the HER2 expression level measured for a sample having a HER2-PLCG1 PPI count>0 in one embodiment (a dotted line indicates an average HER2 expression level, and a red (dark) bar indicates a relapsed sample).
3B is a graph showing the HER3 expression level measured for a sample with a HER2-PLCG1 PPI count>0 in one embodiment (the line represents the mean value of the HER3 expression level, and the dark bar (arrow) represents the relapsed sample).
4A is a graph showing the EGFR expression level measured for 73 samples (the red line (2) is the mean value of the EGFR expression level of all samples, and the green line (1) is the mean value of the EGFR expression level of all samples + standard deviation (SD) : Standard Deviation), hatched bars indicate recurrence patients).
4B is a graph showing the HER2 expression level measured for 73 samples (red line (1) is the mean value of HER2 expression level of all samples, green line (2) is the mean value of HER2 expression level of all samples + standard deviation (SD) : Standard Deviation), hatched bars indicate recurrence patients).
4C is a graph showing the HER3 expression level measured for 73 samples (red line (1) is the mean value of HER3 expression level of all samples, green line (2) is the mean value of HER3 expression level of all samples + standard deviation (SD) : Standard Deviation), hatched bars indicate recurrence patients).
5 is a graph showing the HER2-PLCG1 PPI count measured for a sample having a HER2 expression level equal to or greater than an average value among 73 samples.
6 is a graph showing the results of ROC curve analysis.
7 is a graph showing the results of measuring the HER2 level of a patient whose HER2 level is determined to be 0 or 1 (HER2(-)) by the conventional IHC analysis method by the method for measuring the HER2 level provided herein.
8 is a result of measuring the difference in HER2 expression between a recurrent patient group (R) and a non-recurring patient group (NR) in which breast cancer actually recurred in the sample of the 72 breast cancer patient group of Example 11 by the method for measuring the HER2 level provided herein. is a graph showing
9 shows a patient group classified as a high risk group because the HER2 expression level is higher than the reference level as measured in Example 12 (high risk; dotted line) and a patient group classified as a low risk group because the HER2 expression level is lower than the reference level (low risk; solid line) It is a graph showing the result of analyzing the trend in the survival rate (Disease Free Survival).
Figure 10 shows the high risk group (high risk) and low risk group (low risk) classification results and recurrence rates for 138 breast cancer samples.
11 shows a patient group classified as a high risk group because the HER2 expression level is higher than the reference level as measured in Example 14 (high risk; dotted line) and a patient group classified as a low risk group because the HER2 expression level is lower than the reference level (low risk; solid line) It is a graph showing the result of analyzing the trend in the survival rate (Disease Free Survival).
12 schematically shows a method for measuring HER2 levels provided herein.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

Example 1: Preparation of breast cancer samples

After 72 months of follow-up of 73 breast cancer patients diagnosed as ER (estrogen receptor)/PR (progesterone receptor) positive and HER2 negative (stage 0 or 1 samples in HER2 IHC test; ER/PR+, HER2-), 18 of them were followed up. Recurrence of breast cancer was observed in 10 patients. Breast cancer tissue samples isolated from the 73 ER/PR+ and HER2- patients (provided by Samsung Seoul Hospital) were prepared (see Table 1).

Sample TC (%) Sample TC (%) Sample TC (%) 40 80 24 70 68 70 47 80 25 70 78 70 52 80 26 70 One 60 53 80 27 70 3 60 62 80 33 70 4 60 63 80 35 70 5 60 73 80 37 70 10 60 79 80 42 70 14 60 6 70 43 70 17 60 7 70 45 70 21 60 9 70 46 70 22 60 11 70 51 70 28 60 16 70 55 70 29 60 19 70 61 70 39 60 48 60 SPBCA69 80 44 70 50 60 SPBCA70 70 60 70 54 60 SPBCA71 70 80 70 58 60 SPBCA72 80 66 60 59 60 SPBCA73 70 70 60 20 70 67 70 72 60 74 60 SPBCA74 70 75 60 SPBCA75 60 76 60 SPBCA76 70 77 60 SPBCA77 95 8 50 SPBCA78 70 12 50 SPBCA79 90 31 50 SPBCA80 80 69 50 SPBCA81 70

(In the table above, samples indicated in bold are samples obtained from patients with recurrent breast cancer, TC: Tumor contents (%): ratio of cancer cells/normal cells in the tissue)

The prepared breast cancer tissue was prepared in an ^{amount of about 20 mm 3} , but it may be larger than this.

The prepared tissue is placed on a metal mortar pre-soaked in liquid nitrogen, and crushed into a powder using a hard material such as metal. At this time, it is important to maintain liquid nitrogen so that the tissue does not dissolve. Through this, the surface area per unit volume was increased so that the chemical reaction by the surfactant in the dissolution buffer could occur as efficiently as possible.

The ready-tumor tissue 20 mm ³ was dissolved per buffer (50 mM HEPES-NaOH (pH 7.4), 1% (v / v) Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% (v / v) glycerol, About 200 uL of 1% (v/v) protease inhibitor cocktail (Sigma, P8340) and 1% (v/v) tyrosine phosphatase inhibitor cocktail (Sigma, P5726)) were added, and the rotation was continued for 1 hour in a refrigerator at 4°C. and reacted.

After reaction for 1 hour, centrifugation was performed for 10 minutes at 15,000 g and 4° C. for the reaction. After that, the submerged portion (pellet) was discarded, and only the supernatant was taken and stored until use for the next test.

Example 2: Second protein preparation

In this example, a process of preparing a labeled second protein by attaching a fluorescent protein to a protein that interacts with the first protein (EGFR, HER2, HER3) included in the tissue lysate prepared in Example 1 is exemplified.

The second protein exemplified in this example is summarized in Table 2 below:

protein Accession number label Expression vector (where to get it) PLC-gamma-SH2 A nucleic acid molecule encoding amino acids 545-765 in PLCg (NM_013187.1) or amino acids 540-765 in NP_002651.2 is cloned and used eGFP pEGFP-C1 vector (clontech) Grb2 Cloning the entire sequence of Grb2 (NP_002077.1) PI3K Cloning the entire sequence of Human PI3K regulatory subunit alpha isoform 1 (NP_852664.1)

Expression vectors for each of the second proteins listed in Table 2 were injected into HEK293 cells (ATCC) and cultured to express the second protein. After culturing for 24 hours, the cells were collected, distributed in an appropriate amount, and stored at -80°C. The cell lysate (50 mM HEPES-NaOH (pH 7.4), 1% (v/v) Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) v) protease inhibitor cocktail (Sigma, P8340) and 1% (v/v) tyrosine phosphatase inhibitor cocktail (Sigma, P5726)) were put. Since a high concentration of surfactant (Triton X-100) may interfere with protein interaction, first, 60 uL of lysate was added to ^{5x10 5 cells or cells thereon.}

After dissolving all the cells in the obtained reaction mixture that were aggregated through pipetting, they were reacted for a total of 30 minutes while kept in a cold block (0-4° C.) placed on ice. At this time, a process of physically mixing was performed through periodic pipetting at 10-minute intervals so that the dissolution reaction by the surfactant occurred actively.

After 30 minutes of reaction, centrifugation was performed (10 min, 15,000 g, 4° C.). The sunken part was discarded (pellet) and only aqueous solution (supernatant) was transferred to a new tube for testing.

60 uL of the obtained aqueous solution was taken and added to 140 uL of dilution buffer (50 mM HEPES-NaOH, pH 7.4, 150 mM NaCl). In this way, a final solution of the second protein containing 0.3% Triton X-100 can be obtained. The concentration of the fluorescent protein attached to the second protein was measured using a Fluoremeter. As a result, the concentration of the three second proteins used was determined to be in the range of approximately 400 to 1000 nM.

Example 3: Preparation of Substrate

After washing the coverslip well with tertiary distilled water, it was washed with piranha solution (sulfuric acid: hydrogen peroxide = 2:1 ~ 3:1 (v/v)). Coating was carried out by sequentially treating the washed coverslip surface with a PEG mixture containing aminopropylsilane, polyethylene glycol (PEG), and biotin-PEG in a ratio of 100:3. The ratio of Biotin-PEG may be 100:3 or more.

After 2 hours of reaction, after washing with tertiary distilled water, the PEG-coated side was kept in contact at -20°C until use.

In the following test, the prepared coverslip and an acrylic substrate were used, and the coverslip and the acrylic substrate were thawed before the test and assembled, or after the PEG coating was completed, the prepared coverslip and the acrylic substrate were assembled in advance and stored at -20°C in the assembled form. Thawed before use.

Example 4: Imaging of protein-protein interaction between the first protein and the second protein

Neutravidin (Thermo, A2666), which is an Avidin-based protein, was added to the prepared substrate at a concentration of 0.1 mg/ml. After reacting at room temperature for 10 minutes, put the substrate in a container containing about 10 ml of dilution buffer containing 0.1% Triton X-100 and shake it about 30 times. Shaking in a direction parallel to the surface of the coverslip is good for cleaning effect. The substrate was washed using this. After that, the substrate was turned over for the next material injection, and all the solution in the well was shaken off.

An antibody against the first protein as a target was added to the prepared substrate. At this time, the antibody used was prepared in the form of a biotin conjugated form. The concentration of the antibody can be appropriately adjusted according to the affinity (dissociation constant, KD) of the antibody-antigen, and in this example, about 2 ug/ml was used, and the reaction time was about 10 minutes. If an antibody to which biotin is not conjugated is used, the antibody of the first protein may be attached using a secondary antibody.

Antibodies to the first protein used at this time are summarized in Table 3 below:

first protein antibody Where to get antibodies EGFR anti-EGFR antibody MS-378-B0, Thermo HER2 anti-HER2 antibody BMS120BT, Thermo HER3 anti-HER3 antibody BAM348, R&D systems

The antibody-treated substrate was washed using the method used for washing neutravidin previously. Prior to the prepared substrate, the tissue lysate containing the first protein prepared in Example 1 was added. In the case of an antigen-antibody reaction, the reaction time was set to about 15 minutes because the antigen-antibody reaction could continue to increase up to 15 minutes and the antigen-antibody reaction efficiency could be lowered compared to the time when it exceeds 15 minutes. After the reaction, the substrate is washed in the same manner as in the cleaning method performed above.

Thereafter, the second protein solution obtained in Example 2 was added to the substrate. The concentration of the second protein in the first protein lysate used was set to a value between 1-50 nM (about 30 nM) based on the fluorescent protein. When the second protein concentration is greater than 100 nM, background noise becomes large in the fluorescence microscope, which interferes with accurate measurement of the fluorescence signal.

The substrate was immobilized on a fluorescence microscope and imaged to obtain data for each of the first protein/second protein. In order to increase the accuracy of the results, images were obtained from a total of 10 different locations under the same conditions, and the averaged values were used as representative values.

Example 5: Protein complex (PPI complex) analysis

Protein complexes were analyzed based on the toolkit provided by the Matlab program (provided by MathWorks).

The fluorescence image obtained in Example 4 was stored in a 16-bit unsigned integer format. The fluorescence signal was obtained from enhanced green fluorescent protein (eGFP), and the wavelength of the laser was set to 488 nm to observe the signal, and the laser power was adjusted to 2 mW to maintain the emission time of the eGFP for about 11 seconds. Among all frames (10 frames), the first frame was discarded, and images of 3 frames (7th-9th frames) were averaged to generate one image. The following procedure was performed by acquiring images of dogs. The reason for discarding the first 6 frames is to select and use a section in which an unnecessary signal (autofluorescence) generated on the surface of an empty substrate disappears and the eGFP signal is maintained. The selected section may vary depending on the imaging condition/equipment construction state. In this example, using an EMCCD (Electron-multiplying charge-coupled device; Andor iXon Ultra 897 EX2 (DU-897U-CS0-EXF)) camera, the exposure time per frame is set to 0.1 second, and the EMCCD gain value is set to 40. Fluorescence images were obtained.

To remove the noise, the following procedure was performed for each frame:

(a) The first start is from the upper left corner. One frame consists of 512x512 pixels. Median values were obtained from 11x11 pixels, 11 to the right and 11 to the bottom, based on the reference pixel, and the median value was subtracted from the value of the reference pixel [(Intensity_pixel)-(medianIntensity_11x11neighborhood)]. The above procedure was performed for all pixels of 512x512. Pepper & salt noise was removed through median filtering.

(b) Gaussian smoothing of sigma=0.7 and size=5x5 was performed on the processed image above to smooth the image.

(c) Threshold was set. Threshold makes all the pixel values whose pixel intensity is below the threshold in the entire image as the threshold value (using the algorithm to find the local maximum in Matlab toolkit). Through this, it is possible to remove a local maximum that is not created by the fluorescence signal in the image. The threshold value used in the imaging conditions of this embodiment is 70.

The signal from the fluorescent protein is generated in the form of localized point spread function (PSF), and the number of PPI complexes (biological values) between the first protein and the second protein for which the number of PSFs (physical value) is to be measured. value). The PSF value was converted to a biological value, the PPI complex value, through the following process:

(a) The location of the local maximum was obtained (eg, i-th row, j-th column pixel). As described above, single-molecule fluorescence signals are formed by gathering at a specific location among 512x512 pixels (approximately 5x5 pixel size under current observation equipment, 1 pixel = 0.167 micrometers). This can be obtained using the toolkit provided in Matlab.

(b) A process of determining whether the local maximum obtained in (a) is actually generated from the PSF is performed. First, the minimum intensity value of the local maximum was defined, and only the case greater than this minimum value among the obtained local maximums was used for analysis. The minimum value used in this embodiment is 75, and this value may vary depending on the laser power/exposure time/equipment construction situation. After fetching 5x5 pixel information based on the finally obtained local maximum coordinates, the center of brightness was obtained from this 5x5 pixel (centroid of intensity). At this time, if the obtained brightness center deviated by more than 0.5 pixel with respect to the existing local maximum coordinates (2D symmetry with respect to the PSF shape disappears), it was judged as an abnormal fluorescence signal and excluded from the analysis.

(c) Only PSFs that passed all conditions were finally selected to obtain the coordinates and total number. The total number of PSFs obtained at this time becomes the number of PPI complexes.

The above process was performed for all files photographed under the same conditions to obtain the number of PSFs, and then the average and standard deviation were obtained by combining them. This value finally becomes a value indicating the number of PPI complexes under specific conditions (refer to FIG. 1).

Example 6. Correlation between PPI and breast cancer recurrence

EGFR, HER2, and HER3, which are known to be overexpressed in breast cancer, are selected as the first protein, and PLCG1 (PLC-gamma1), PI3K, and Grb2, which are their representative sub-signaling pathway proteins, are selected as the second protein, and the PPI (protein-protein interaction) was measured, and the correlation between the result and breast cancer recurrence was confirmed.

After measuring the PPI complex between HER2-PLCG1 and EGFR-Grb2 by the method described in Example 5, the PPI complex value when only the second protein was treated without sample treatment on the substrate prepared in Example 3 The subtracted value (PPI count) was obtained:

PPI count = [PPI complex value measured in the sample] - [PPI complex value measured in untreated sample]

The obtained results are shown in Table 4 and FIG. 2a (HER2-PLCG1 PPI count), 2b (EGFR-Grb2 PPI count):

sample number Recurrence HER2-PLCG1 EGFR-Grb2 (comparative example) #One 24.18182 17.04 #10 68.81819 33.84 #11 2.9 #12 63.01818 77.40 #14 35.9 82.44 #16 O -21.70909 -24.30 #17 78.9 133.14 #19 1.5 #20 -36.5 -72.60 #21 16.17273 28.00 #22 138.8 72.84 #24 -17.58182 -60.43 #25 -29.7 -65.10 #26 -37.7 #27 -5.12727 #28 87.4 61.45 #29 32.2 29.10 #3 4.95455 15.84 #31 68.8 131.00 #33 -15.4 -42.57 #35 -14.03636 #37 -8.9 #39 17.56364 59.40 #4 2.25455 5.54 #40 40.01818 6.90 #42 -10.3 5.00 #43 9.87273 #45 -13.72727 #46 -28.4 13.60 #47 41.78333 5.20 #48 92.1 23.10 #5 12.36364 8.36 #50 425.4 22.60 #51 5.81818 25.36 #52 45.2 20.00 #53 29.10909 25.67 #54 O -21.2 -0.40 #55 11.1 #58 31.9 60.70 #59 O 222.35 89.62 #6 -2.2 17.40 #61 -24.98182 -5.20 #62 0.3 8.18 #63 O 30.10909 77.30 #66 18.01667 -2.00 #67 -11.8 13.40 #68 -8.09091 #69 67.8 76.18 #7 -38.4 -63.30 #70 26.50909 5.60 #72 75.76667 52.70 #73 O -11.4 11.80 #74 -9.49091 3.12 #75 91.6 73.00 #76 6.5 #77 12.39091 #78 -0.4 31.60 #79 -2.63636 18.46 #8 49.9 63.64 #9 20.2 -4.10 SP69 O 30 16.90 SP70 O 383.4 61.90 SP71 O 114.8 66.70 SP72 O 22.6 18.70 SP73 O 43.3 82.70 SP74 O 32.4 SP75 O -16.7 -5.91 SP76 O -14.1 61.30 SP77 O 27.6 -4.20 SP78 O -31 -14.70 SP79 O 50.92727 1.10 SP80 O -0.8 68.40 SP81 O 64.74545 50.00

(In the table above, samples marked with 'O' for relapse means relapsed samples) As shown in Table 4 and FIGS. 2A to 2B (dark bars indicate relapse patients), PPI count is 0 or less ( That is, in the case of HER2-PLCG1, the non-recurring patient ratio is the highest among the samples in which the PPI value in the sample is lower than the noise value or background signal value of the measurement system (ie, the PPI complex value measured when the sample is not processed). The HER2-PLCG1 PPI count was selected as a biomarker for breast cancer recurrence because the sample with a PPI count greater than zero had the highest proportion of relapse patients.

Example 7. HER2 - PLCG1 PPI when count>0 HER2 or HER3 Correlation between expression level and breast cancer recurrence

In Example 6, the HER2 (biomarker) expression level was measured for samples having a HER2-PLCG1 PPI count greater than 0 (HER2-PLCG1 PPI count>0), and the expression level of HER2 or HER3 and breast cancer recurrence were determined. Correlation was tested.

The expression levels of HER2, HER3, and EGFR in the samples were measured as follows (refer to the upper right figure of FIG. 1 ). Specifically, the step of immobilizing the antibody on the prepared substrate proceeds in the same manner as for PPI measurement.

To measure the amount of HER2, HER3, and EGFR, the first protein is fixed on the substrate by injecting the same sample concentration to the substrate through Bradford or other protein quantification method. Thereafter, a washing process is performed, and a detection antibody for detecting the first protein is injected (Table 5). After 10 minutes of reaction, a secondary antibody capable of measuring the detection antibody (Rabbit IgG-Cy3 or Alexa 488 conjugation) is injected, and after 10 minutes of reaction, a final washing process is performed.

first protein antibody Where to get antibodies EGFR anti-EGFR antibody #4267, CST HER2 anti-HER2 antibody MA5-15050, Thermo HER3 anti-HER3 antibody #4791, CST Rabbit IgG anti-rabbit IgG antibody 111-165-046, Jackson

After the reaction is completed, the amount of each first protein is quantified by measuring the number of single-molecule PSFs in the same manner as in the PPI complex measurement through a total reflection microscope.

The HER2 expression level in the obtained HER2-PLCG1 PPI count>0 samples is shown in FIG. 3A (in FIG. 3A, the dotted line represents the mean value of the HER2 expression level, and the dark bar represents the relapsed sample).

As can be seen from FIG. 3A , among the 14 samples with a higher HER2 expression level than the average value of the HER2 expression level of the HER2-PLCG1 PPI count>0 samples, 8 samples had recurrence of breast cancer, and 6 samples had no recurrence. On the other hand, among the 33 samples with lower HER2 expression levels than the average value, 3 samples showed recurrence of breast cancer and 30 samples showed no recurrence, indicating that the breast cancer recurrence rate in samples with a higher HER2 expression level than the average value ([ 8/14]*100=57%) was significantly higher than the breast cancer recurrence rate ([3/33]*100=9%) in samples with lower-than-average HER2 expression levels.

These results show that, after the HER2-PLCG1 PPI count is measured, when the HER2 expression level is continuously measured for a sample with a HER2-PLCG1 PPI count>0, when the HER2 expression level is greater than or equal to the average value of the samples with a HER2-PLCG1 PPI count>0 This result confirms that the probability of recurrence of breast cancer is significantly increased.

The following indicators were measured based on the obtained HER2 (biomarker) expression level, and are shown in Table 6:

Recurrence Patient Judgment Reliability (Positive Prediction Value, PPV; %) = [(number of relapsed samples among samples with biomarker expression level greater than mean or mean + SD (standard deviation))/(biomarker expression level is mean or mean + total number of samples greater than SD)]*100;

Negative Prediction Value (NPV; %) = [(number of non-recurrent samples among samples with biomarker expression levels below the mean or mean+SD)/(of samples with biomarker expression levels below the mean or mean+SD) total number)]*100;

Recurrent patient detection rate (Sensitivity; %) = [(number of relapsed samples among samples with biomarker expression level above the mean or mean + SD)/(total number of recurrent samples)]*100;

Non-recurring patient exclusion rate (Specificity; %) = [(Number of non-recurring samples among samples with biomarker expression levels below the mean or mean+SD)/(total number of non-recurring samples)]*100;

relapse number of relapse samples Number of non-recurring samples Biomarker (HER2) expression level Above average (434.5) 8 6 Below average (434.5) 3 30 Reliability of recurrent patient determination (PPV) 57% Non-recurring patient determination reliability (NPV) 91% Recurrent patient determination rate (Sensitivity) 73% Non-recurrence exclusion rate (Specificity) 83%

In addition, in the same manner as above, in Example 6, the HER3 (biomarker) expression level was measured for samples having a HER2-PLCG1 PPI count greater than 0 (HER2-PLCG1 PPI count>0), and the HER3 expression level and Correlation with breast cancer recurrence was tested.

The HER3 expression level in the samples with the measured HER2-PLCG1 PPI count>0 is shown in FIG. 3B (the line represents the mean value of the HER3 expression level, and the dark bar (arrow) represents the relapsed sample), and the relapse patient determination reliability, ratio Indices of recurrent patient determination reliability, recurrent patient determination rate, and non-recurrent patient exclusion rate were measured, and are shown in Table 7:

relapse number of relapse samples Number of non-recurring samples Biomarker (HER3) expression level above average 7 9 below average 4 27 Reliability of recurrent patient determination (PPV) 44% Non-recurring patient determination reliability (NPV) 87% Recurrent patient determination rate (Sensitivity) 64% Non-recurrence exclusion rate (Specificity) 75%

As can be seen in Tables 6 and 7 above, when evaluating and measuring the expression level of HER2 or HER3 as a biomarker for samples with HER2-PLCG1 PPI count>0, generally excellent results in the diagnostic index of breast cancer recurrence In particular, when the expression level of HER2 was measured and evaluated for samples with HER2-PLCG1 PPI count > 0, relapse patient determination reliability, non-relapse patient determination reliability, relapse patient determination rate, and non-relapse patient exclusion rate showed better results in all the evaluated indices of

Example 8. biomarker After measuring the expression level HER2 - PLCG1 PPI Measure to confirm correlation with breast cancer recurrence

The correlation between the HER2-PLCG1 PPI count measurement and the biomarker (HER2 or HER3) expression level measurement was tested for correlation with breast cancer recurrence when the order was changed.

More specifically, for all samples (73) prepared in Example 1, referring to the method of Example 7, after measuring the expression levels of HER2 and HER3 as biomarkers, respectively, the sample whose expression level is above the average value was selected. Thereafter, with respect to the selected sample, the HER2-PLCG1 PPI count is measured with reference to the method of Example 6, and a sample with a HER2-PLCG1 PPI count > 0 is selected, and patient determination reliability, non-recurrence patient determination reliability, and recurrent patient determination are made. rate, and indicators of non-recurring patient exclusion rates were measured.

The obtained results are shown in FIG. 5 (HER2), Table 8 and Table 9.

Results of HER2-PLCG1 PPI count measurement for samples with HER3 expression levels above the average value relapse number of relapse samples Number of non-recurring samples biomarker
(HER2-PLCG1 PPI count) >0 7 7 <0 4 13 Reliability of recurrent patient determination (PPV) 50.0% Non-recurring patient determination reliability (NPV) 76.5% Recurrent patient determination rate (Sensitivity) 63.6% Non-recurrence exclusion rate (Specificity) 65.0%

Results of HER2-PLCG1 PPI count measurement for samples with HER2 expression levels above the average value relapse number of relapse samples Number of non-recurring samples biomarker
(HER2-PLCG1 PPI count) >0 8 7 <0 0 7 Reliability of recurrent patient determination (PPV) 53.3% Non-recurring patient determination reliability (NPV) 100.0% Recurrent patient determination rate (Sensitivity) 100.0% Non-recurrence exclusion rate (Specificity) 50.0%

Example 9. Assessment of diagnostic performance

The breast cancer recurrence diagnostic performance of the results obtained in Examples 6 to 8 was evaluated through a Likelihood ratios in diagnostic testing and Youden's index, and the results are shown in Table 10 below:

How to measure line color Best cutoffs Youden's
index Sensitivity Specificity LR+ LR- HER2 Gray (a) 434.51 0.26 44.4% 81.8% 2.44 0.68 EGFR Black (b) 196.70 0.21 50.0% 70.9% 1.72 0.71 HER3 Orange (c) 239.52 0.25 61.1% 63.6% 1.68 0.61 HER2 PPI>0, HER2 Red (d) 434.51 0.62 72.7% 88.9% 6.55 0.31 HER2 PPI>0, EGFR Cyan (e) 196.70 0.23 45.5% 77.8% 2.05 0.70 HER2 PPI>0, HER3 Magenta(f) 240.10 0.41 63.6% 77.8% 2.86 0.47 HER2>mean, HER2 PPI Navy (g) 16.85 0.63 100.0% 62.5% 2.67 0.00 HER3>mean, HER2 PPI Olive (h) 21.40 0.44 63.6% 80.0% 3.18 0.46 EGFR>mean, HER2 PPI Brown (i) 21.40 0.33 60.0% 72.7% 2.20 0.55

(LR+, Possibility Ratio: [Ratio of actual recurrence samples among samples determined to be positive (+) for recurrence / ratio of samples to actual recurrence among samples determined to be negative (-)]), the higher the number, the better the prediction performance, LR-, Negative probability ratio: [Ratio of non-recurring samples among samples determined to be recurrent positive (+) / Ratio of actual non-recurring samples among samples determined to be recurrent negative (-)], the smaller the number, the better the prediction performance;

Youden's index: Sensitivity + Specificity - 1; When the Sensitivity and Specificity are expressed in %, a value divided by 100 is used), the closer to 1, the better the diagnostic performance;

Best cutoff for HER2 PPI>0, HER2 level:

- 434.5

- Sensitivity : 72.7%

- Specificity : 88.9%

In addition, the results of analyzing the results of Table 8 by ROC curve analysis are shown in Table 11 and FIG. 6 (ROC curve) below.

How to measure line color Area Error p-value 95% LCL 95% UCL HER2 Gray 0.604 0.069 0.187 0.468 0.740 EGFR Black 0.574 0.071 0.350 0.435 0.712 HER3 Orange 0.568 0.077 0.391 0.417 0.718 HER2 PPI>0, HER2 Red 0.770 0.068 0.007 0.637 0.903 HER2 PPI>0, EGFR Cyan 0.551 0.094 0.615 0.366 0.735 HER2 PPI>0, HER3 magenta 0.611 0.095 0.269 0.425 0.797 HER2>mean, HER2 PPI Navy 0.719 0.138 0.086 0.449 0.988 HER3>mean, HER2 PPI Olive 0.709 0.096 0.058 0.521 0.898 EGFR>mean, HER2 PPI Brown 0.636 0.100 0.223 0.440 0.833

ROC curve analysis is a widely used analysis method in the diagnostic field. The larger the area under the graph, the better the performance, and the smaller the asymptotic probability (p-value), the more statistically significant the results. Through this, it can be objectively demonstrated that the method provided herein has superior performance compared to other methods.

The ROC curve shows the results of plotting LR+ in Table 10 for various thresholds (LR+ becomes the y-axis/x-axis value at a specific location).

Area is the value obtained by integrating the area under the graph, standard error is the error caused by inaccuracy that occurs when connecting the points forming the graph, and Asymptotic Probability (p-value) is the value of the recurrent patient when a sample is accidentally selected. It is an indicator showing how much the difference is from the probability of being discovered. The smaller the number, the lower the probability, meaning that it is farther from chance, that is, the diagnosis accuracy is high.

UCL and LCL represent the range of Area values assuming 95% confidence, meaning that the probability that the correct Area value is between LCL<Area<UCL is 95%.

The measurement methods shown in Tables 10 and 11 are summarized in Table 12 below:

How to measure Explanation HER2 By measuring the HER2 expression level of the sample, the correlation with the recurrence of breast cancer was confirmed with reference to Example 7 (see FIG. 4b ) EGFR By measuring the EGFR expression level of the sample, check the correlation with the recurrence of breast cancer with reference to Example 7 (see Fig. 4a) HER3 By measuring the HER3 expression level of the sample, the correlation with the recurrence of breast cancer was confirmed with reference to Example 7 (see FIG. 4c ) HER2 PPI>0, HER2 Example 4-7 HER2 PPI>0, EGFR After performing Example 4-6, the EGFR expression level was measured and the correlation with the recurrence of breast cancer was confirmed with reference to Example 7 HER2 PPI>0, HER3 After performing Example 4-6, HER3 expression level was measured and correlation with breast cancer recurrence was confirmed with reference to Example 7 HER2>mean, HER2 PPI See Example 8 and FIG. 5 HER3>mean, HER2 PPI see Example 8 EGFR>mean, HER2 PPI HER2-PLCG1 PPI count measurement with reference to Example 6 for a sample with an EGFR expression level greater than or equal to the average value

As shown in Tables 10, 11, and 6 above, the method of evaluation by performing HER2-PLCG1 PPI count measurement and HER2 or HER3 expression level measurement is the most excellent in both of the tested indicators, in particular, Sensitivity and Specificity. results can be obtained,

The quantitative diagnostic performance index Youden's index (Sensitivity+Specificity-1) and the positive/negative probability ratio (LR+/LR-) were also found to be excellent, confirming excellent performance in predicting breast cancer recurrence.

In particular, in the method of evaluating HER2-PLCG PPI count measurement and HER2 expression level measurement in any order, the Youden's index exceeded 0.5 and was closest to 1, and these results indicate that the method predicts breast cancer recurrence. This means that it shows very good performance.

When the above results are taken together, the method for evaluating the HER2-PLCG PPI count and the HER2 or HER3 expression level, especially the HER2 expression level measurement provided herein, is the recurrent patient determination reliability, the non-recurring patient determination reliability, and the recurrent patient. In addition to showing excellent results in all indicators of the determination rate and non-recurring patient exclusion rate, it can be said that it shows excellent breast cancer recurrence prediction performance in the results evaluated by the positive/negative probability ratio and Youden's index.

Example 10: Measurement of HER2 expression level in samples obtained from breast cancer patients

Neutravidin, an Avidin-based protein, was added to a substrate coated with a PEG mixture containing polyethylene glycol (PEG) and biotin-PEG. After washing the substrate, an antibody against HER2 protein was treated. At this time, the antibody used was prepared in the form of a biotin conjugated form. The concentration of the antibody may be appropriately adjusted according to the affinity (dissociation constant, KD) of the antibody-antigen. When using an antibody to which biotin is not conjugated, an antibody of the HER2 protein may be attached using a secondary antibody.

After washing the antibody-treated substrate, the prepared breast cancer tissue lysate was added. In the case of an antigen-antibody reaction, the reaction time was set to about 15 minutes because the antigen-antibody reaction could continue to increase up to 15 minutes and the antigen-antibody reaction efficiency could be lowered compared to the time when it exceeds 15 minutes.

Thereafter, the HER2 antibody to which the fluorescent protein was attached was added. The HER2 antibody used in this case is characterized in that it acts on a site different from that of the antibody attached to the substrate. Data were obtained by measuring the fluorescence signal of the fluorescent protein by fixing the substrate on a fluorescence microscope and imaging. In order to increase the accuracy of the results, images were acquired from a total of 10 different locations under the same conditions, and the averaged values were used as representative values.

Example 11: Prediction of breast cancer recurrence compared to a reference value

As a method for setting reference values, ER (estrogen receptor)/PR (progesterone receptor) positive (level 2 or 3 in IHC test) and HER2 negative (step 0 or 1 in HER2 IHC test) (ER/PR+, HER2- ), prepared a breast cancer tissue sample from 72 breast cancer patients, and obtained the fluorescence signal value according to the HER2 expression level as data in the same manner as in Example 10. The average value or average value±standard deviation (1*SD) of 72 measured values was set as a reference value.

As the number of samples of a patient (ie, N value) increases and data is accumulated, the reference value may be slightly different, but the accuracy may be higher. When the signal value measured in Example 10 is equal to or greater than the reference value, the breast cancer prognosis is bad, and when the signal value is lower than the reference value, the breast cancer prognosis is predicted to be good.

The obtained results are shown in FIGS. 7 and 8 .

7 is a graph showing the results of measuring the HER2 level of a patient whose HER2 level is determined to be 0 or 1 (HER2(-)) by the conventional IHC analysis method by the HER2 level measurement method provided herein. Even in patients classified as (-) (HER2 0 or 1), when measured by the HER2 level measurement method provided herein, there is a large difference in HER2 level, and even among patients classified as HER2(-), patients with a significant level of HER2 expression shows that it may exist.

8 is a result of measuring the difference in HER2 expression between the recurrent patient group (R) and the non-recurring patient group (NR) in which breast cancer actually recurred in the sample of the 72 breast cancer patient group in this Example by the HER2 level measurement method provided herein. is a graph showing

Example 12: Analysis of correlation between prediction of breast cancer recurrence and actual survival

With respect to 72 samples used to set the reference value in Example 11, the measured signal value of the sample was divided into a high risk group equal to or greater than the reference value and a low risk group lower than the reference value. There were 9 high risk groups and 63 row risk groups. The correlation between the prediction of breast cancer recurrence and the actual survival rate (Disease Free Survival) is shown in FIG. In FIG. 9 , the dotted line shows the survival rate of the high-risk group, and the solid line shows the survival rate of the low-risk group, respectively. As shown in FIG. 9 , it can be seen that the survival rate of the high-risk group decreases significantly faster than the low-risk group, thereby proving the effectiveness of the recurrence diagnosis technique performed in this example.

Example 13: HER2 target therapy applied patient group selection step

In Examples 10 and 11, the high-risk group greater than or equal to the reference value has a relatively high HER2 expression level, and may be selected as a patient group effective for HER2-targeted therapy. The HER2 target therapy is a drug targeting the HER2 protein, typically Herceptin (trastuzumab), and other HER2-TKI or pan-HER TKI, but is not limited thereto. It can be used as a companion diagnostic method for HER2 targeted therapy as it can be used for HER2 targeted therapy for patients who were previously classified as HER2 (-) and could not receive targeted therapy.

Example 14: Expansion of breast cancer samples and prediction of breast cancer recurrence

In the breast cancer tissue samples of 72 breast cancer patients used in Example 11, ER (estrogen receptor)/PR (progesterone receptor) positive (step 2 or 3 in IHC test) and HER2 negative (step 0 or 1 in HER2 IHC test) ( By adding breast cancer tissue samples from 66 breast cancer patients diagnosed with ER/PR+, HER2-), a total of 138 breast cancer tissue samples were prepared.

With reference to Examples 10 and 11, the fluorescence signal value according to the HER2 expression level was measured for the 138 breast cancer samples prepared above, and the average value or the average value of the fluorescence signal measurement values according to the HER2 expression level of the 138 breast cancer samples ± The standard deviation (1*SD) was set as the reference value.

Among the 138 breast cancer samples, the group in which the fluorescence signal value by the HER2 expression level was higher than the reference value was classified as a high risk group, and the group lower than the reference value was classified as a low risk group, and then recurrence was analyzed. .

The results of analyzing the HER2 expression level of the obtained 138 breast cancer samples, their classification into high risk and low risk groups, and recurrence rates are shown in FIG. 10 . In addition, the correlation between the prediction of breast cancer recurrence and the actual survival rate (Disease Free Survival) was measured and shown in FIG. As shown in FIG. 10 , breast cancer recurred in patients derived from 28 samples out of a total of 138 samples, and the overall recurrence rate was 20.3%. Specifically, 17 samples out of 107 samples classified as low-risk groups The recurrence rate of the low-risk group was 15.8% in the low-risk group due to recurrence of breast cancer in patients derived from It was found to be about twice as high, which shows that the p-value by the log-rank test is at a statistically significant level of about 0.014, which shows that it is possible to predict the high risk of recurrence. In addition, as shown in FIG. 11, it can be seen that the survival rate of the high-risk group (dotted line) sharply decreases significantly faster than that of the low-risk group (solid line), thereby demonstrating the effectiveness of the relapse diagnosis technique performed in this example. can be

Claims (13)

(I) measuring a protein-protein interaction between HER2 and PLC-gamma protein in a biological sample isolated from a breast cancer patient, and
(II) measuring the expression level of HER2 or HER3 in a biological sample isolated from a breast cancer patient
A method of providing information in predicting breast cancer recurrence, comprising a. According to claim 1,
Proceed in the order of steps (I) and (II),
After step (I),
(Ia) further comprising comparing the protein-protein interaction level between the HER2 and PLC-gamma protein of the biological sample measured in step (I) with a reference protein-protein interaction level,
In step (II), when the level of protein-protein interaction between HER2 and PLC-gamma protein in the biological sample measured in step (I) is higher than or equal to the reference protein-protein interaction level, as a result of the comparison of step (Ia), It is performed by measuring the expression level of HER2 or HER3 in the biological sample,
The reference protein-protein interaction level is a protein-protein interaction level measured in a state in which a biological sample or a biological sample and PLC-gamma protein is not added,
How to inform the prediction of breast cancer recurrence. 3. The method of claim 2,
After step (II), (II-a) further comprising the step of comparing the expression level of HER2 or HER3 measured in step (II) with a reference expression level,
How to inform the prediction of breast cancer recurrence. According to claim 1,
Proceed in the order of steps (II) and (I),
After step (II),
(II-a) further comprising comparing the expression level of HER2 or HER3 measured in step (II) with a reference expression level,
In step (I), when the expression level of HER2 or HER3 measured in step (II) is greater than or equal to the reference expression level as a result of the comparison of step (II-a), the protein between HER2 and PLC-gamma protein in the biological sample - carried out by measuring protein interactions,
wherein the reference expression level is an average value of the expression level of HER2 or HER3 in samples isolated from two or more breast cancer patients with or without the patient from which the biological sample was isolated,
How to inform the prediction of breast cancer recurrence. 5. The method of claim 4,
After step (I),
(Ia) further comprising comparing the protein-protein interaction level between the HER2 and PLC-gamma protein of the biological sample measured in step (I) with a reference protein-protein interaction level,
How to inform the prediction of breast cancer recurrence. According to any one of claims 1 to 5, wherein the breast cancer,
(1) HER2 negative (-) breast cancer,
(2) breast cancer in which the estrogen receptor (ER), the progesterone receptor (PR), or both are positive (+), or
(3) breast cancer having both the characteristics of (1) and (2),
How to inform the prediction of breast cancer recurrence. 7. The method of claim 6,
The HER2-negative (-) breast cancer is a score determined by immunohistochemical staining (IHC) using a HER2-specific antibody (IHC score) is 1 or less,
The breast cancer in which the estrogen receptor (ER), the progesterone receptor (PR), or both of them are positive (+) is a score determined by immunohistochemical staining (IHC) using an ER-specific antibody, a PR-specific antibody, or both. (IHC score) is 2 or more,
How to inform the prediction of breast cancer recurrence. According to any one of claims 1 to 5, wherein the protein-protein interaction between the HER2 and PLC-gamma protein,
preparing a substrate on which the HER2 protein in the sample is immobilized by applying a biological sample isolated from a breast cancer patient to a substrate including a material that specifically binds to the HER2 protein on the surface;
reacting the prepared HER2 protein by adding the labeled LC-gamma protein to the immobilized substrate, and
Measuring a signal generated from the obtained reactant
A method of providing information to predict breast cancer recurrence, which is measured by delete delete delete delete delete

Images