WO2020080891A1

WO2020080891A1 - Method for predicting recurrence of breast cancer

Info

Publication number: WO2020080891A1
Application number: PCT/KR2019/013761
Authority: WO
Inventors: 이홍원; 이정언; 김상민; 최희준; 이슬
Original assignee: 주식회사 프로티나; 사회복지법인 삼성생명공익재단
Priority date: 2018-10-19
Filing date: 2019-10-18
Publication date: 2020-04-23
Also published as: KR20200044706A; KR102317202B1

Abstract

Disclosed is a method and a kit for predicting (or diagnosing) a breast cancer prognosis (for example, whether the cancer is recurring) through the analysis of protein-protein interactions from among proteins associated with breast cancer and/or the measurement of expression levels of the proteins associated with breast cancer, or for providing information for the prediction (or diagnosis). In addition, the present invention relates to: a method for predicting (or diagnosing) a breast cancer prognosis or whether breast cancer is recurring or for selecting a patient suitable for HER2 targeted therapy, through HER2 protein expression level; or a method for providing information for the prediction (or diagnosis) or selection.

Description

How to predict breast cancer recurrence

Predict (or diagnose) breast cancer prognosis (eg, recurrence) or provide information to the prediction (or diagnosis) by analyzing protein-protein interactions between proteins associated with breast cancer and / or measuring the expression level of the protein associated with breast cancer It relates to a method and kit for. In addition, the present invention relates to a method for predicting (or diagnosing) prognosis or recurrence of breast cancer through HER2 protein expression level, or selecting a patient suitable for HER2 target treatment, or a method for providing information on the prediction (or diagnosis) or screening.

Until recently, personalized diagnosis and treatment according to the individual of the disease have been mainly focused on genomic profiling. However, the cause of certain diseases, including cancer, is due to the abnormal activity of the cells that make up the body, more specifically the abnormal expression of various proteins that make up and regulate the cells (eg, overexpression) and / or abnormal interactions between proteins. However, there is a problem that is difficult to understand only by observation through genetic analysis. Accurate analysis of protein-protein interactions not only allows you to understand how intracellular signaling networks are changing, but also predicts the progress and properties of individual cancers, providing relevant and useful information for the treatment. It is expected to be possible.

In existing breast cancer diagnosis, 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 by HER2 (-) through IHC scoring.

In general, in the case of HER2 (+), target anticancer therapy and chemotherapy are prescribed, and in the case of HER2 (-), hormone therapy is prescribed. In the case of HER2 (-), a high-risk group with a high probability of recurrence or metastasis is prescribed in combination with chemotherapy. In the diagnosis of recurrence of existing HER2 (-) breast cancer, it is classified into a high-risk group and a low-risk group by predicting the metastasis or recurrence using RT-PCR or Microarray for a specific plurality of genes.

The IHC scoring technique used for diagnosis of HER2 (+/-) is based on the degree of staining. Interpretation of results may vary depending on the tester, and the accuracy of the analytical method is low. Significant levels of HER2 can be included, which can impede proper treatment of the patient.

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

In the present specification, in the protein diagnosis method, a technique for measuring the protein level more precisely than the conventional IHC scoring technique is provided, and the high-risk group with a high probability of recurrence is more effectively classified.

One example is a method of predicting breast cancer prognosis or recurrence (or diagnosis), or a method of providing information to a breast cancer prognosis or recurrence prediction (or diagnosis), comprising measuring the HER2 protein expression level of a biological sample isolated from a breast cancer patient. Gives

More specifically, the method,

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

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

It may include.

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

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

The method, after the step (ii), (iii-a) if the HER2 protein expression level (signal value) measured in step (i) is higher than the reference expression level (reference value), the reference expression level (reference value) ), As compared with the case where the biological sample is isolated, confirming that the breast cancer prognosis is poor or the likelihood 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), compared to the case where the reference expression level (reference value) or higher, the prognosis of breast cancer in the patient with the biological sample isolated is good, or It may further include the step of confirming that the probability of recurrence is low.

In another example, the method comprises monitoring the prognosis or recurrence of breast cancer after step (iii-a), or preventing and / or treating breast cancer recurrence (eg, preventing or treating breast cancer recurrence). Treatment, drug administration, radiation, and / or surgical operations, etc.). In other embodiments, the method may further include monitoring the prognosis or recurrence of breast cancer after step (iii-b). The step of monitoring the prognosis or recurrence of breast cancer may be one or more of the above-described methods for predicting prognosis or recurrence of breast cancer.

Another example provides a patient selection method suitable for HER2 target treatment, or a method of providing information for patient selection, comprising measuring the HER2 protein expression level of a biological sample isolated from a breast cancer patient.

More specifically, the method,

(ii) the HER2 protein expression level measured in step (i) above the reference expression level (e.g., the level of HER2 protein expression in a sample isolated from two or more breast cancer patients with or without the biological sample is isolated) Step)

It may include.

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

In another example, the method may further include, after step (iii), (iv) performing a HER2 target treatment on the breast cancer patient selected in step (iii).

Another example is

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

(ii) the HER2 protein expression level measured in step (i) above the reference expression level (e.g., the level of HER2 protein expression in a sample isolated from two or more breast cancer patients with or without the biological sample is isolated) Average value),

(iii) selecting a breast cancer patient derived from a biological sample having a HER2 protein expression level measured in step (i) above a reference expression level as a patient suitable for HER2 target treatment,

(iv) performing HER2 target treatment on the breast cancer patient selected in step (iii) above

It provides a method for treating breast cancer, including.

Another example is

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

Other examples include predicting (or diagnosing) the prognosis of breast cancer, including measuring protein-protein interactions between HER2 and PLC-gamma proteins in biological samples isolated from breast cancer patients and measuring the levels of HER2 and / or HER3. A) method, or a method of providing information to predict (or diagnose) breast cancer prognosis.

More specifically, the method,

(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

It may include.

In one embodiment, the method,

(I) measuring the protein-protein interaction between HER2 and PLC-gamma protein in biological samples isolated from breast cancer patients,

(Ia) The level of protein-protein interaction between HER2 and PLC-gamma protein of the biological sample measured in step (I) above is a reference protein-protein interaction level (e.g., protein-protein interaction when no biological sample is 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 the reference protein-protein interaction level, the biological sample Measuring the expression level of HER2 and / or HER3

It may include.

In another embodiment, the method comprises:

(Ia) The level of protein-protein interaction between HER2 and PLC-gamma protein of the biological sample measured in step (I) above is a reference protein-protein interaction level (e.g., protein-protein interaction when no biological sample is added) Step of comparing action levels),

(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, and

(II-a) The expression level of HER2 and / or HER3 measured in step (II) above is a reference expression level (e.g., in a sample separated from two or more breast cancer patients with or without the isolated patient) Comparing with the mean value of the expression level of HER2 or HER3)

It may include.

In another embodiment, the method comprises:

(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 in

(II-a) The expression level of HER2 and / or HER3 measured in step (II) above is a reference expression level (e.g., in a sample separated from two or more breast cancer patients with or without the isolated patient) Comparing with the mean value of the expression level of HER2 or HER3), and

(II-b) When the expression level of HER2 and / or HER3 measured in step (II) is above the reference expression level, compared with the case where it is lower than the reference expression level, the prognosis of breast cancer in a patient with the biological sample is poor Things, such as confirming that the likelihood of 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, or higher than the reference expression level Comparing the case, the step of confirming that the biological sample has a good breast cancer prognosis of the patient, for example, the likelihood of recurrence of breast cancer is low

It 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,

(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 higher than the reference expression level, the protein-protein between HER2 and PLC-gamma protein of the biological sample Steps to measure interaction

It may include.

In another embodiment, the method comprises:

(II-a) The expression level of HER2 and / or HER3 measured in step (II) above is a reference expression level (e.g., in a sample separated from two or more breast cancer patients with or without the isolated patient) Comparing with the average value of the expression level of HER2 or HER3),

(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 higher than the reference expression level, the protein-protein between HER2 and PLC-gamma protein of the biological sample Measuring the interaction, and

(Ia) The level of protein-protein interaction between HER2 and PLC-gamma protein of the biological sample measured in step (I) above is a reference protein-protein interaction level (e.g., protein-protein interaction when no biological sample is added) Step of comparing action levels)

It may include.

In another embodiment, the method comprises:

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

(Ib) When the level of protein-protein interaction between HER2 and PLC-gamma protein measured in step (I) is greater than the reference protein-protein interaction level, compared to a case where the reference protein-protein interaction level is lower than or equal to A protein-protein between the HER2 and the PLC-gamma protein measured in step (I) above, and / or (Ic) confirming that the patient with the biological sample isolated has a poor breast cancer prognosis, such as a high probability of recurrence of breast cancer. When the level of interaction is below the reference protein-protein interaction level, compared to the case where the reference protein-protein interaction level is greater than that of the patient with the biological sample isolated, the prognosis of breast cancer is good, for example, the probability of breast cancer recurrence is low. Steps to Confirm

It may include.

In another example, the method comprises monitoring the prognosis of breast cancer, or preventing and / or treating breast cancer recurrence (e.g., for the prevention or treatment of breast cancer recurrence) after steps (II-b) or (Ib) above. , Drug administration, radiation, and / or surgical operations, etc.). In other embodiments, the method may further include monitoring the prognosis of breast cancer after step (II-c) or (I-c). The step of monitoring the prognosis of breast cancer may be performed one or more times of the method for predicting breast cancer prognosis described above.

Another example is

The reaction part between HER2 and PLC-gamma protein containing a substrate on which a substance specifically binding to HER2 is immobilized on a surface,

HER2 and / or HER3 expression level measurement unit, or

All of these

It provides a kit for predicting or diagnosing a prognosis (eg, recurrence) of breast cancer. The kit adds a signal detection means capable of detecting a signal (eg, a fluorescent signal, etc.) by a reaction between the HER2 and the PLC-gamma protein, and / or a PLC-gamma protein labeled with a signal material (eg, fluorescent material). It can contain as.

In the present specification, a protein associated with breast cancer, such as HER2 and / or HER3 protein expression level and / or protein between HER2 and PLC-gamma protein, is used in a biohaled sample isolated from a breast cancer patient using protein interaction analysis technology. -Provide diagnostic techniques useful for establishing appropriate treatment strategies 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, eg, breast cancer symptom changes (improvement, alleviation, treatment (removal), or anti- breast cancer treatment), or Exacerbation, etc.), breast cancer recurrence, etc., and having a good prognosis means that breast cancer symptoms are improved, alleviated, or treated (removed), and / or the likelihood of recurrence of breast cancer is low, and the prognosis is bad. The term may mean that breast cancer symptoms worsen or the likelihood of breast cancer recurring is high.

As used herein, "breast cancer recurrence" means that breast cancer recurs after it has healed, and breast cancer is cured (eg, breast cancer tissue by anticancer treatment such as surgical surgery, drug administration, irradiation, etc.) It means that the tumor of the same type reoccurs at the same site (primary recurrence) or at another site (metastatic recurrence) after removal, disappearance, and / or reduction of the equivalent degree to disappearance). Breast cancer recurrences include: ① direct relapses in which tumor cells that have not been completely eliminated (eg, local recurrence and metastatic recurrence); And ② after the tumor cells are completely removed, there are indirect recurrences in which the same type of tumor is newly generated. Recurrence of breast cancer generally occurs in malignant tumors, but does not exclude cases in benign tumors (eg, local recurrence). In the present specification, "predicting or diagnosing breast cancer recurrence" may mean confirming, determining, or determining in advance whether or not the possibility of recurrence of breast cancer in a patient in whom breast cancer has healed. Typically, the likelihood of breast cancer recurrence 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 treatment” can be selected from all therapeutic means that specifically inactivate, inhibit expression or reduce level (concentration), and / or eliminate HER2 proteins, eg For example, administering a drug targeting a HER2 protein and / or gene (e.g., specifically inhibiting a HER2 protein and / or gene) (e.g., a small molecule compound, a HER2 target protein (e.g., antibody, etc.)) can do.

As used herein, "patient suitable for HER2 target treatment" may mean a patient predicted that HER2 target treatment described above will be effective, and that the HER2 target treatment is effective is desired when applying HER2 target treatment. It can mean that it is possible to obtain an anti-cancer effect against breast cancer (e.g., decrease in cancer tissue, disappearance, and / or decrease in relapse rate, etc.).

More specifically, the method,

It may include.

The step of measuring the expression level of HER2 protein in step (i) is to administer (add) a HER2 binding agent with a label attached to a biological sample isolated from a breast cancer patient, and to measure a signal generated from the label in a near-field region. It can be performed by the steps.

In another example, the method for predicting prognosis or recurrence of breast cancer includes, 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, irradiation, and / or surgical operations, etc.). In other embodiments, the method may further include monitoring the prognosis or recurrence of breast cancer after step (iii-b). The step of monitoring the prognosis or recurrence of breast cancer may be one or more of the above-described methods for predicting prognosis or recurrence of breast cancer.

More specifically, the method,

It may include.

Another example is

(ii) the level of HER2 protein expression measured in step (i) above the reference expression level (e.g., the level of HER2 protein expression in a sample isolated from two or more breast cancer patients with or without the biological sample is isolated) Average value),

It provides a method for treating breast cancer, including.

Another example is

Provided is a kit for predicting or diagnosing breast cancer prognosis or recurrence), or a patient selection kit suitable for treatment of HER2 targets, including a HER2 protein expression level measuring unit comprising a substrate immobilized on a surface of HER2 protein. The kit may include a signal generated by binding (reaction) between a labeled HER2 binding substance (e.g., a fluorescently labeled anti-HER2 antibody, etc.), and / or binding (reaction) between the HER2 protein and a binding substance, specifically binding to the HER2 protein. , A fluorescent signal, etc.) may be further included. 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 the mean value ± standard deviation (SD) of the expression level of HER2 in two or more reference breast cancer samples. It can mean something. 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 sample to be tested. In one example, 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 undergone breast cancer treatment (eg, surgical surgery 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 may 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 commonly used protein expression level measurement methods (eg, ELISA, Western blotting, Immunohistochemistry (IHC), etc.). For example, the expression level of the HER2 protein can be measured and / or quantified by a single-molecule pull down assay. The single-molecule pull down assay includes applying a sample containing the target protein to a substrate on which the target protein (HER2) is directly or indirectly (eg, through an antibody, etc.) immobilized; Reacting by treating a HER2 binding substance to which a labeling substance (eg, a fluorescent substance) is attached; And detecting (measuring) measuring the generated signal (eg, a fluorescence signal) in a near-field region, and measuring a target protein expression level using the detected (measured) signal (eg, a fluorescence signal) and And / or quantifying. The near field may be determined according to the generated signal (fluorescence signal). The HER2 binding substance is (a) a labeled (eg, fluorescent substance attached) or unlabeled captured protein (prey protein) and / or (b) HER2 protein or the captured protein that specifically binds to the HER2 protein (bait protein). (Eg, an unlabeled capture protein) may include a label (eg, attached to a fluorescent material) that specifically binds (eg, an antibody).

In one embodiment, the reference expression level of the HER2 is the total amount of protein in the sample, the amount of protein expressed by the number of fluorescent spots measured in the area of 45 (um) * 90 (um), as measured by the method provided herein. The standardized value to be 0.5mg / 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 does not work.

All steps performed in the methods provided herein can be performed in vitro isolated from a living body (such as the human body).

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

More accurate breast cancer prognosis (e.g., breast cancer recurrence) by performing protein-protein interaction measurements between HER2 and the proteins that interact with it in addition to measuring the HER2 protein expression level, and / or HER3 protein expression level, as described above. Prediction is possible.

Another example is predicting (or diagnosing) breast cancer prognosis, comprising measuring protein-protein interactions between HER2 and PLC-gamma proteins in a biological sample isolated from a breast cancer patient and measuring the level of HER2 and / or HER3. Methods, or methods of providing information to predict (or diagnose) breast cancer prognosis.

More specifically, the method,

It may include. The steps (I) and (II) can be performed in any order.

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

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 (e.g., noise value or background signal value in the measurement system), the It was confirmed that the prognosis of breast cancer in patients with biological samples isolated is poor (eg, the probability of breast cancer recurrence is high).

Thus, the method comprises, after the step of 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). Comparing the level to a reference protein-protein interaction level can further include (in this case, prior to the comparing step, further comprising measuring a reference protein-protein interaction level), Optionally, in addition, (Ib) if 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 reference protein-protein interaction Compared to the case below the level, the step of confirming that the biological prognosis of the patient whose biological sample is isolated has a poor prognosis, such as a high probability of recurrence of breast cancer. Can include horizontal. At this time, the step of measuring the expression level of HER2 and / or HER3 in step (II) is a protein between HER2 and PLC-gamma protein of the biological sample measured in step (I), as a result of comparison in step (Ia). It can be characterized in that it is performed when the level of protein interaction is greater than the reference protein-protein interaction level.

The reference protein-protein interaction level in steps (I) and / or (Ia) refers to a noise value or a background signal value in the measurement system, without adding a biological sample or a biological sample and PLC-gamma protein. Protein-protein interaction levels measured at.

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

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

In another example, the method may include sequentially including the steps (I) and (II) in the order of (II) to (I). After the step of 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 And optionally, in addition, (II-b) when the expression level of HER2 and / or HER3 measured in step (II) above is equal to or higher than the reference expression level, compared to a case where the expression level is lower than the reference expression level. , The step of confirming that the biological prognosis of the patient from which the biological sample is isolated has a poor prognosis of breast cancer, for example, 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, compared with the case where the expression level is higher than the reference expression level, the step of confirming that the biological prognosis of the patient from which the biological sample is isolated has a good prognosis, for example, the probability of breast cancer recurrence is low It can hamhal. At this time, the step of measuring the protein-protein interaction of step (I) performed after step (II) is a comparison result of step (II-a), HER2 of the biological sample measured in step (II), and And / or may be characterized in that it is performed when the expression level of HER3 is higher than the reference expression level.

At this time, after the step of 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 include a step of comparing with the protein-protein interaction level, and optionally, in addition, (Ib) the protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I) above. When the reference protein-protein interaction level is greater than the reference protein-protein interaction level, the biological sample is identified as having a poor breast cancer prognosis, for example, a high probability of recurrence It may further include a step. As described above, the step of measuring the protein-protein interaction of step (I) is a comparison result of 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 larger than the reference expression level.

In steps (II), (II-a), (II-b), and (II-c), the reference expression level is the average value or the mean value ± standard deviation of the expression level 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 sample to be tested. In one example, 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 undergone breast cancer treatment (eg, surgical surgery treatment), about 5 years or 6 years after the treatment It may be a sample capable of confirming the prognosis of breast cancer (eg, whether or not recurrence) over the years. In addition, the reference breast cancer sample means two or more reference breast cancer samples having a protein-protein interaction level between HER2 and a PLC-gamma protein measured by the method described in step (I) above a reference protein-protein interaction level. You can. The reference expression level may be databased and applied to the methods provided herein.

In another embodiment, if the prognosis of breast cancer is breast cancer recurrence, the reference expression level is, for the two or more reference breast cancer samples described above,

-"Sensitivity (%) = [((Biomarker expression level exceeded the reference expression level) sample recurring sample count) / recurring sample total count) / (recurring sample count) when both steps (I) and (II) were performed" ] * 100) + specificity (Specificity (%) = [(number of non-recurring samples among samples where the biomarker expression level is below the reference expression level) / (total number of non-recurring samples)] * 100)-value obtained by 1 "( Youden's index) is at least 0.5, at least 0.51, at least 0.52, at least 0.53, at least 0.54, at least 0.55, or at least 0.56, and / or

-When both of the above steps (I) and (II) are sequentially performed, the "positive probability ratio ([Recurrence positive (+) sample rate of actual recurring samples / Recurring negative (-) sample" is the actual recurrence among samples Sample rate])-A value obtained with a negative likelihood ratio ([Ratio of actual non-recurring samples among samples with positive recurrence (+) / Ratio of actual non-recurring samples among samples with negative recurrence (-)]) "is 2.5 or higher, 2.75 Or higher, 3 or higher, 3.25 or higher, 3.5 or higher, 3.75 or higher, or 4 or higher

It may be to have a value (expression level) to make this. The above values can be applied to the method provided in this specification after being databased.

In one embodiment, the reference expression level is the amount of protein measured by a conventional protein level measuring means, and in the case of HER2, the number of fluorescent spots measured in the region of 45 (um) * 90 (um) when measured by immunofluorescence method. The standardized value of the expressed protein amount so that the total protein content in the sample is 0.5 mg / ml is 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 level 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 sample to be tested. In one example, 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 undergone breast cancer treatment (eg, surgical surgery 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 may be databased and applied to the methods provided herein.

As used herein, the expression level of a protein may refer to the amount of protein in a sample, which is a conventional method clearly known to those skilled in the art to which the present invention pertains (eg, ELISA, Western blotting, Immunohistochemistry (IHC), etc.) can be measured and / or quantified.

In one example, the method,

(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 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

It may include.

In another example, the method,

(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 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 above the reference expression level, compared with the case where it is lower than the reference expression level, the prognosis of breast cancer in a patient with the biological sample is poor (E.g., confirming that the likelihood of recurrence of breast cancer is high, and / or (II-c), if the expression level of HER2 and / or HER3 measured in step (II) is lower than the reference expression level, the reference expression level Compared to the above case, the step of confirming that the biological sample has a good prognosis for breast cancer, for example, the likelihood of recurrence of breast cancer is low.

It may include.

In another embodiment, the method comprises:

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

It may include.

In another example, the method,

It may include.

In another embodiment, the method comprises monitoring the prognosis of breast cancer, or preventing and / or treating breast cancer recurrence (e.g., preventing or treating breast cancer recurrence) after steps (II-b) or (Ib). Treatment, drug administration, radiation, and / or surgical operations, etc.). In other embodiments, the method may further include monitoring the prognosis of breast cancer after steps II-c) or (I-c). The step of monitoring the prognosis of breast cancer may be performed one or more times of the method for predicting breast cancer prognosis described above.

Another example is after the above method for predicting (or diagnosing) the prognosis of breast cancer, or after providing a method for predicting (or diagnosing) the prognosis for breast cancer (e.g., after step (II-b) or (Ib)) or above. Drug administration, radiation, and / or surgical surgery for the prevention or treatment of breast cancer recurrence in patients identified as having a poor breast cancer prognosis in (II-b) or (Ib), such as a high probability of breast cancer recurrence It provides a method for preventing and / or treating breast cancer or breast cancer recurrence, comprising the step of performing. In the methods described herein, the biological sample is cancer tissue, cancer cells, or cultures thereof isolated from a breast cancer patient. You can. The breast cancer patient has breast cancer or has been diagnosed with breast cancer, a patient receiving breast cancer treatment (e.g., surgical treatment, drug administration, anti-cancer treatment such as radiation), and / or breast cancer is cured (e.g., surgical operation, drug It may be a patient who has been removed from breast cancer tissue by an anti-cancer treatment such as administration or radiation, and disappeared, and / or reduced to an extent equivalent to disappearance. 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. The breast cancer is (1) HER2 negative (-) breast cancer, (2) hormone receptor (HR; estrogen receptor (ER) and / or progesterone receptor (PR)) positive (+) breast cancer, or (3) the It may be breast cancer having both the characteristics of (1) and (2). The breast cancer patient may be a patient determined (or diagnosed or judged) as a breast cancer patient determined (confirmed) with HER2 (-) and / or determined (confirmed) with ER / PR (+) and HER2 (-). have. The methods described herein are HER2 negative (-), and / or hormone receptor (HR) (e.g., estrogen receptor (ER) and / or progesterone receptor (PR)) positive (+) confirmed (diagnosed, determined) 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, PR specific antibody, and / or HER2 specific antibody. It can be determined by the score determined by the histochemical staining method (IHC) (for example, when determining the IHC score (0-3), ER / PR (+) is the IHC score (intensity) ≥ 2 (2 or 3), HER2 (-) may be the case of IHC score (intensity) ≤1 (0, or 1+)).

In addition to performing step (i) above, the sample may further include the step of measuring the expression level of (a) estrogen receptor (ER) and / or (b) PD-1 and / or CD3e. In this case, the prognosis for breast cancer is poor or the likelihood of breast cancer recurring is higher than that in (a) high ER expression levels in the sample and / or (b) low PD-1 and / or CD3e expression levels. You can decide to be high. Evaluation of the ER expression level and the expression level of PD-1 and / or CD3e (high or low) may be evaluated in comparison with a reference expression level, and the reference expression level is, for example, as analyzed by IHC, The level of ER expression in HER2 (-) cells (eg, SKBR3), or the expression level of PD-1 and / or CD3e, but is not limited thereto.

In determining the ER / PR level, conventional anti-ER antibodies (e.g. clone 6F11, 1: 200 dilution, Novocastra, Bond-Max system) and anti-PgR antibodies (e.g. clone 16, 1: 800 dilution, Novocastra , Immunostaining using Bond-Max system), scoring the stained nuclei (not cytoplasm) (typically following IHC scoring; intensity score (0-3) and proportion score (0-5)), the value It can be judged to be positive when it appears to be equal to or greater than 1% of cancer cells, and negative when it is lower than 1% of cancer cells, but is not limited thereto. In addition, in determining the level of HER2, anti-HER2 antibodies (e.g., 4B5, Ventana, BenchMark XT) are immunostained, and the stained nuclei (not cytoplasm) are scored (generally follow IHC scoring), followed by It 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 mean physical and / or chemical bonding or complex formation between a first protein and a second protein, eg, It may be measured by one or more of factors such as binding frequency, bonding strength (strength), and bonding 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 the direct interaction (binding) between them, but also other proteins in the middle (of the signaling pathway). It may also include interactions (binding) mediated by proteins located between the first protein and the second protein. The protein-protein interaction herein can be a single molecule reaction (a reaction between a first protein in one molecule and a second protein in one molecule).

As used herein, HER2, estrogen receptor (ER), PD-1, CD3e, EGFR, HER3, and / or PLC-gamma are each independently from mammals such as primates such as humans, monkeys, and rodents such as mice and rats. It 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 or the like. EGFR is a GenBank Accession Nos. It may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1, etc. HER3 is GenBank Accession No. It may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_001982 or 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, rodents such as mice and rats, and 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 an SH2 domain thereof (Src homology 2 domain: N-terminal SH2 domain and one or more of C-terminal SH domains; for example, NP_037319. It may be the 545th to 765th amino acid sequence region of 1 or the 540th or 545th to 765th amino acid sequence region of NP_002651.2, etc.).

Determination of the expression level of HER2 protein, estrogen receptor (ER), PD-1, and / or CD3e (hereinafter referred to as the first protein) may be performed by protein-protein interaction, for example, the first It may be measured by one or more of factors such as the frequency of physical and / or chemical bonding (interaction), binding strength (strength), and binding time between the protein and the second protein that binds it. In addition, the binding between the first protein and the second protein is not only a direct bond 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 the third protein) May be). The protein-protein interaction herein can be a single molecule reaction (a reaction between a first protein in one molecule and a second protein in one molecule).

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 substance specifically binding to the first protein is immobilized It can be quantified by immobilizing the first protein on a substrate by treatment, adding a labeled detecting antibody (second protein), and measuring the protein-protein interaction between them. The protein-protein interaction can be quantified by measuring the signal generated by the labeling substance attached to the second protein.

The step of measuring the protein-protein interaction between the first protein (HER2) and the second protein (e.g., HER2 binding antibody) performed in the methods provided herein comprises ex vivo or extracellular ( in vitro ). 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 is ex vivo or extracellular () for isolated cells or tissues. in vitro ).

Hereinafter, in the description of the step of measuring the protein-protein interaction, the first protein means HER2 and the second protein means 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) The first protein is fixed by adding a biological sample isolated from a breast cancer patient containing the first protein to a substrate containing a substance (eg, anti-HER2 antibody) that specifically binds the first protein to the surface. Preparing the substrate;

(2) reacting by adding the labeled second protein (bound material bound) to the substrate on which the prepared first protein is fixed; And

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

Optionally, measuring the protein-protein interaction may include, in addition to 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). It can contain.

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

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

The steps are described in more detail below:

Step (1): first protein immobilized substrate preparation step

The step (1) is a step of preparing a substrate to which the first protein is immobilized by adding a biological sample containing the first protein to a substrate containing a substance specifically binding to the first protein.

The first protein may be HER2.

The biological sample is a cell (eg, cancer cell), tissue (eg, cancer tissue) isolated from a breast cancer patient, a lysate, lysate, or extract of the cell or tissue, body fluid (eg, blood (whole blood, plasma, or serum). , Saliva, etc.). When the biological sample is tissue (eg, cancer tissue), it is sufficient if it is at least 125 mm ^{3 in} size for grinding, 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 It can, but is not limited to this. 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, and has ordinary knowledge in the technical field to which this invention belongs It is a value that can be appropriately determined by the ruler. The breast cancer patient may be a mammal such as a human who has been diagnosed with and / or treated for breast cancer, and may be a target for predicting or diagnosing the prognosis (eg, recurrence) of breast cancer.

The substrate may have all materials and / or all structures capable of immobilizing the first protein on the surface (both crystalline or amorphous can be used). In one example, the substrate may be made of a material having a refractive index of light (about 1.3) or higher, which accounts for a large portion of the biomaterial, in consideration of the ease of detection of the 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, and may be obtained from a material selected from the group consisting of glass (refractive index: about 1.52), quartz, etc., but is not limited thereto. The substrate may have any form that is commonly used for observation of biological samples, and may be, for example, a well type, a slide type, a channel type, an array form, a microfluidic chip, a microtube (capillary), etc., but is not limited thereto. It does not work. When observing the fluorescent microscope, it can be observed by mounting the cover glass on the substrate to which the sample is applied, and the material of the cover glass is as described above for the substrate, and the thickness may be less than or equal to the range described for the substrate (for example, refractive index 1.52, It may be 0.17 mm thick, but is not limited to this).

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 specifically binding to the first protein or antigen binding thereof 1 type selected from the group consisting of fragments (e.g., antibody scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2), aptamers (protein or nucleic acid molecules), small molecule compounds, etc. It may be abnormal. At this time, the material that specifically binds to the first protein is a site that does not interfere with the interaction between the first protein and the second protein, that is, the site where the first protein and the second protein interact (bond). It may be binding to the first protein at the non-site.

In one example, the substrate is appropriately surface-modified to include (immobilize) a biological material (eg, antibody, etc.) that specifically binds to the first protein, or specifically to the first protein directly on the surface. The binding material may be fixed. When the substrate is surface-modified, the substrate can be treated (e.g., applied) with any compound having a functional group capable of immobilizing a biological material (e.g., antibody, etc.) that specifically binds to the first protein on one surface. And may be treated with a compound containing a functional group selected from the group consisting of aldehyde groups, carboxyl groups and amine groups. In one embodiment, the compound comprising a functional group selected from the group consisting of the aldehyde group, carboxyl group and amine group is biotin, bovine serum albumin (BSA), biotin-bound bovine serum albumin, polyethylene glycol (polyethylene) glycol; PEG), biotin-linked PEG (polyethylene glycol-biotin; PEG-biotin), polysorbate (eg, Tween20), or the like. The surface-treated substrate may be further treated (eg, applied) by one or more selected from the group consisting of neutravidin, streptavidin, avidin, and the like.

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

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

The second protein may be a HER2 binding antibody.

The labeled second protein may be labeled with a labeling substance that causes the second protein to generate a detectable signal (the labeling substance may be, for example, chemically (eg, covalently or non-covalently), recombinantly, or physically, bound) , It may mean a tag attached form to which a labeling material can be bound. The detectable signal can be selected from all signals (eg, light, radiation, etc.) that can be measured through conventional enzyme reaction, 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, etc. capable of generating the label signal, for example, fluorescent dyes (small molecule compounds; Cyanine, Alex, Dylight, Fluoprobes, etc.) ), Fluorescent protein (e.g., green fluorescent protein (GFP, enhanced GFP), yellow fluorescent protein (YFP), blue fluorescent protein (CFP), blue fluorescent protein (BPF), red fluorescent protein (RPF), etc.) It may be one or more selected from. The tag may be at least one selected from all commonly used types such as His-tag / Ni-NTA. The concentration of use of the labeling material can be appropriately determined within a 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 can be measured by any signal detection means commonly used to detect or measure it (eg, a conventional fluorescence microscope, a fluorescence camera, a fluorescence intensity measurement (quantitative) device, etc.).

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 measuring step (step (3)), the substrate on which the reaction occurred It may further include the step of washing in a conventional manner.

Step (3): Protein-protein interaction measurement step

Step (3) is a step of measuring a signal from the reactant obtained in step (2). The signal measurement is any means capable of detecting (or measuring or confirming) the label signal used in step (2) (for example, a signal measurable through conventional methods such as enzyme reaction, fluorescence, luminescence, or radiation detection). It 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 performed by supplying a light source absorbed by a label material that generates the fluorescence signal, for example, a fluorescence microscope, a fluorescence camera, and / or fluorescence The intensity measurement (quantitative) device or the like can be used to image and / or quantify.

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

If the signal is a fluorescence signal, step (3) (step of measuring protein-protein interaction) is

(a) supplying a light source to the reactant in step (2); And

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

It may include.

The step of supplying the light source in step (a) is a step of supplying a light source to the reactants of the first protein and the second protein obtained in step (2). If such a goal can be achieved, the supply time 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 not limited thereto. It is not.

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

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

In one embodiment, the protein-protein interaction measurement step of step (3) is performed by supplying a light source using a Total Internal Reflection Fluorescence (TIRF) microscope or a confocal microscope, and a fluorescent signal in a conventional manner. It can be performed by observing. In another example, a light source is supplied and a fluorescent signal is imaged by using a fluorescent camera for signal imaging, such as an EMCCD (Electron-multiplying charge-coupled device) camera or a Complementary metal oxide semiconductor (CMOS) camera. And / or quantitation.

In the following, the case where 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, for example:

A) The substrate of step (1) or step (2) is mounted on a total reflection microscope. In the total reflection microscope, the supply position of the light source is usually downward, and depending on the type of the total reflection microscope, the fluorescent signal is placed on the substrate (in this case, from bottom to top, the light source supply, the substrate, the lens, or the substrate, the light source supply, the lens Can be in 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) Can be observed.

B) The light source may be a laser, and the intensity of the light source is about 0.5 mW to about 5 mW, about 0.5 mW to about 4.5 mW, about 0.5 mW to about 4 mW, about 0.5 mW to about 3.5 mW, 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 It may be from 1.5 mW to about 3.5 mW, from about 1.5 mW to about 3 mW, from about 1.5 mW to about 2.5 mW, or from about 1.5 mW to about 2 mW, such as about 2 mW, and the wavelength of the light source is fluorescence as described above. It may be appropriately selected according to the signal or the labeling material used, and / or the configuration of the equipment (for example, a strong light source may be used when an attenuation filter is used).

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

The photographing (or imaging) of the fluorescence signal may be performed simultaneously with the supply of the light source or within the signal generation maintenance time in consideration of the fluorescence signal generation maintenance time (light emission time, lifetime) of the labeling substance.

When shooting (or imaging) a fluorescent signal with a fluorescent camera (eg, an EMCCD camera), the exposure time per frame, laser power, camera gain value, and total shooting frame can be appropriately adjusted. For example, the shorter the exposure time per frame, the less signals are accumulated in one frame, and to compensate for this, the laser power may be increased or the sensitivity of the fluorescent camera may be increased. In one example, the exposure time per frame is about 0.001 seconds to about 5 seconds, about 0.001 seconds to about 3 seconds, about 0.001 seconds to about 2 seconds, about 0.001 seconds to about 1 second, about 0.001 seconds to about 0.5 seconds, About 0.001 seconds to about 0.3 seconds, about 0.001 seconds to about 0.1 seconds, about 0.01 seconds to about 5 seconds, about 0.01 seconds to about 3 seconds, about 0.01 seconds to about 2 seconds, about 0.01 seconds 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 seconds to about 0.5 seconds, about 0.05 seconds to about 0.3 seconds, about 0.05 seconds to about 0.1 seconds, about 0.07 seconds to about 5 seconds, about 0. 07 seconds to about 3 seconds, about 0. 07 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, about 0.1 seconds to about 3 seconds, about 0.1 seconds to about 2 seconds, about 0.1 seconds to about 1 second, about 0.1 second to about 0.5 second, or about 0.1 second to about 0.3 second, such as about 0.1 second, but is not limited thereto.

For example, when an EMCCD camera is used, 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 a gain value. As the set gain value increases, the number of electrons generated per photon increases, so the sensitivity of the EMCCD increases and the background noise increases, so the signal-to-noise ratio is important. In one embodiment, to obtain a good signal-to-noise ratio, the gain value is 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 , It is not limited to this, and may be appropriately selected in consideration of camera sensitivity, life, equipment construction status, noise, test conditions, and the like.

Multiplying the total number of shooting frames by the exposure time gives the total shooting time (exposure time * total number of shooting 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 can be adjusted so that the photographing time proceeds within the emission time.

In order to increase the accuracy of the protein-protein interaction measurement results, one or more of the imaging, such as two or more, three or more, four or more, five or more, seven or more, or ten or more (the upper limit is the Performed on a substrate of one or more channels (determined according to the size and imageable area) or one or more channels including each (each channel includes two or more substrates), to obtain the number of spots (also referred to as PPI complexes) on which a signal appears, or And / or the obtained fluorescence signal can be quantified, which can be viewed as quantifying protein-protein interactions.

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

Step (4): Determining the protein-protein interaction level for the biological sample unit amount and / or the first protein unit amount (optional step)

Step (4) uses the signal measured in step (3) to obtain the 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. It can be a step.

The level of protein-protein interaction for a unit amount of a biological sample and / or first protein is a step for a unit amount of a biological sample and / or a first protein (unit weight or concentration (e.g., 1 ug / ml)) ( It means the signal value (quantified value of signal or signal strength) measured in 3),

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

(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 first protein in the biological sample (i.e., the biological sample and / or the biological sample) It can be obtained by obtaining the amount of the first protein (weight, concentration, or fluorescent signal) in the x-axis, and obtaining the signal value measured in step (3) as the y value to obtain the slope of the graph.

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

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

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

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

The method provided herein may further include the step of normalizing it in order to increase the accuracy of the measurement values obtained in steps (I) and / or (II).

The standardization step measures the HER2 protein expression level in step (i), the protein-protein interaction level in step (I) and / or the measurement of HER2 and / or HER3 protein expression level in step (II) between samples. It may be that the protein amount or concentration is equally adjusted to a certain value. In this case, the total protein amount or concentration for normalization in steps (I) and (II) can be the same or different from each other. In addition, the total protein amount or concentration for the standardization can be arbitrarily determined to be the same between samples.

In the present specification, the expression level of the protein such as HER2, HER3, EGFR or the amount of protein may mean the amount of protein in a biological sample, which can be measured by any conventional method, such as a conventional immunoblotting method (eg , 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 substance specifically binding to the protein is immobilized to immobilize the protein on the substrate, a labeled detection antibody is added thereto, and a signal generated from the label is generated. The protein can be quantified by measurement, but is not limited thereto.

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

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

Another example is

Provided is a kit for predicting or diagnosing breast cancer prognosis or recurrence), or a patient selection kit suitable for treatment of HER2 targets, including a HER2 protein expression level measuring unit comprising a substrate immobilized on a surface of HER2 protein. The kit may include a signal generated by binding (reaction) between a labeled HER2 binding substance (e.g., a fluorescently labeled anti-HER2 antibody, etc.), and / or binding (reaction) between the HER2 protein and a binding substance, specifically binding to the HER2 protein. , A fluorescent signal, etc.) may be further included. In the kit, the HER2 protein expression level measurement unit is a portion where a signal is generated by a reaction between HER2 and a labeled HER2 binding agent, and is a well (eg, multi-well) type, slide type, channel type, array type, microfluidic It may be in the form of a chip, a micro tube (capillary), but is not limited thereto.

Another example is

HER2 and / or HER3 expression level measurement unit, or

All of these

It provides a kit for predicting or diagnosing a prognosis (eg, recurrence) of breast cancer. The kit further includes a signal detection means capable of detecting a signal (eg, a fluorescent signal, etc.) caused by a reaction between a labeled substance (eg, fluorescent substance) labeled PLC-gamma protein and / or the HER2 and a PLC-gamma protein. It can contain. The reaction portion between the HER2 and the PLC-gamma protein may be a well (eg, multi-well) type, slide type, channel type, array type, microfluidic chip, microtube (capillary), but is not limited thereto. The substance specifically binding to the substrate and HER2 is 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 as described in step (2) above.

In the kit provided in the present specification, the signal detection means may be any signal detection means commonly used depending on the signal generated from the labeling substance used. For example, the signal detection means may include a signal stimulation unit and a signal detection unit, and in addition, may further include a signal analysis unit for analyzing (eg, quantitative or imaging) the measured signal. In one example, signal stimulation, signal detection, and signal analysis can each be performed at different sites, or at least two or more of them may be performed simultaneously or sequentially at one site. In one example, when the label is a fluorescent substance, the signal detection means may be selected from all means capable of generating and detecting a fluorescence signal, for example, a fluorescence signal stimulator (eg, light source), and a fluorescence signal It may include a detection unit, and / or a fluorescent signal analysis unit. In one embodiment, the signal detection means includes a total internal fluorescence microscope (Total Internal Reflection Fluorescence (TIRF) microscope) or a confocal microscope (for detecting light sources and fluorescence signals), or in addition, a fluorescence camera, such as EMCCD (Electron A multiplying charge-coupled device (CMOS) camera or a complementary metal oxide semiconductor (CMOS) camera may be additionally included to perform light source supply and imaging and / or quantification of fluorescent signals. The wavelength, intensity, and fluorescence camera measurement conditions of the light source (eg, exposure time per frame, laser power, camera gain value, total shooting frame, etc.) are as described in step (3) above.

The methods provided herein measure the level of expression of a given biomarker and / or test by measuring the PPI between proteins that interact with the biomarker, thereby predicting the prognosis of a breast cancer patient, in particular, whether or not they will relapse in advance. It is expected that it will be useful as a diagnostic technique for personalized treatment of each patient, as it is possible to establish a more effective treatment strategy 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 count measured for 73 samples in one embodiment.

2B is a graph showing EGFR-Grb2 PPI count measured for 73 samples in one embodiment.

Figure 3a is a graph showing the HER2 expression level measured for a sample of HER2-PLCG1 PPI count> 0 in one embodiment (dotted line represents the mean value of HER2 expression level, red (dark) bar represents the recurring sample).

Figure 3b is a graph showing the HER3 expression level measured for a sample of HER2-PLCG1 PPI count> 0 in one embodiment (the line represents the mean value of the HER3 expression level, the dark bar (indicated by the arrow) represents the recurring sample).

Figure 4a is a graph showing the EGFR expression level measured for 73 samples (red line (2) is the average value of the EGFR expression of the entire sample, green line (1) is the average value of the EGFR expression of the entire sample + standard deviation (SD : Standard Deviation), bars marked with hatched lines indicate patients with recurrence).

Figure 4b is a graph showing the HER2 expression level measured for 73 samples (red line (1) is the average value of HER2 expression of the entire sample, green line (2) is the average value of the HER2 expression level of the entire sample + standard deviation (SD : Standard Deviation), bars marked with hatched lines indicate patients with recurrence).

Figure 4c is a graph showing the HER3 expression level measured for 73 samples (red line (1) is the average value of the HER3 expression level of the entire sample, green line (2) is the average value of the HER3 expression level of the entire sample + standard deviation (SD : Standard Deviation), bars marked with hatched lines indicate patients with recurrence).

FIG. 5 is a graph showing HER2-PLCG1 PPI counts measured for samples having a HER2 expression level of an average value or higher among 73 samples.

6 is a graph showing the results of ROC curve analysis.

FIG. 7 is a graph showing the results of measuring the HER2 level of a patient determined to have a HER2 level of 0 or 1 (HER2 (-)) by the conventional IHC analysis method using the HER2 level measurement method provided herein.

8 is a result of measuring the difference in HER2 expression in the sample of 72 breast cancer patient groups of Example 11 in the recurrence patient group (R) in which breast cancer actually relapsed and the non-relapse patient group (NR) in which recurrence of breast cancer was measured by the HER2 level measurement method provided herein. It is a graph showing.

FIG. 9 shows a result of measurement in Example 12, where the HER2 expression level is higher than the reference level, so the patient group classified as a high-risk group (high risk; dashed line) and the HER2 expression level is lower than the reference level, and classified as a low-risk group (low risk; solid line). It is a graph showing the results of analyzing the trend of (Disease Free Survival).

10 shows the high risk and low risk classification results and relapse rates for 138 breast cancer samples.

FIG. 11 shows a result of measurement in Example 14, in which the HER2 expression level is higher than the reference level, so that the patient group classified as a high-risk group (high risk; dashed line) and the HER2 expression level is lower than the reference level and is classified as a low-risk group (low risk; solid line). It is a graph showing the results of analyzing the trend of (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.

실시예 1: 유방암 샘플의 준비Example 1: Preparation of breast cancer samples

73 patients with breast cancer who were determined to be positive for ER (estrogen receptor) / PR (progesterone receptor) and HER2 negative (a 0 or 1 stage sample in the HER2 IHC test; ER / PR +, HER2-) were followed up for 72 months, 18 of them It was observed that breast cancer was relapsed in two patients. A breast cancer tissue sample (provided by Samsung Seoul Hospital) isolated from the 73 ER / PR + and HER2- patients was prepared (see Table 1).

(In the table above, the sample in bold is a sample obtained from a patient with recurrence of breast cancer,

TC: Tumor contents (%): ratio of cancer cells / general cells in the tissue)

The prepared breast cancer tissue was prepared in an amount of about 20 mm ³ , but may be larger.

The prepared tissue is placed on a metal mortar pre-soaked in liquid nitrogen, and crushed into a powder form 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 widened, so that the chemical reaction by the surfactant in the lysis buffer could occur as efficiently as possible.

Lysis buffer per 20 mm ^{3 of} the prepared tumor tissue (50 mM HEPES-NaOH (pH 7.4), 1% (v / v) Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% (v / v) glycerol, Add about 200 uL of 1% (v / v) protease inhibitor cocktail (Sigma, P8340) and 1% (v / v) tyrosine phosphatase inhibitor cocktail (Sigma, P5726), and continue to rotate for 1 hour in a 4 ℃ refrigerator And reacted.

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

실시예 2: 제2 단백질 준비Example 2: Second protein preparation

In this embodiment, a process of preparing a labeled second protein by attaching a fluorescent protein to a protein interacting with the first protein (EGFR, HER2, HER3) contained in the tissue lysate prepared in Example 1 is illustrated.

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

The expression vector for each of the second proteins listed in Table 2 was injected into HEK293 cells (ATCC) and cultured to express the second protein. After incubation for 24 hours, the cells were collected and then distributed in an appropriate amount and stored at -80 ° C.

Lysate in the cells (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) Protease inhibitor cocktail (Sigma, P8340) and 1% (v / v) tyrosine phosphatase inhibitor cocktail (Sigma, P5726)) were added. Since high concentration of surfactant (Triton X-100) may interfere with protein interaction, 60 uL of lysate was first added to 5x10 ⁵ cells or cells on it.

After releasing all the cells in the obtained reaction mixture, which were clustered through pipetting, they were reacted for a total of 30 minutes while being stored in a cold block (0-4 ° C) on ice. At this time, a physical mixing process was performed through periodic pipetting at intervals of 10 minutes, so that the dissolution reaction by the surfactant was actively performed.

After the reaction for 30 minutes, centrifugation was performed (10 min, 15,000 g, 4 ° C.). The submerged portion was discarded (pellet), and the aqueous solution (supernatant) was transferred to a new tube for experimentation.

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 solution of a second protein containing 0.3% Triton X-100 can be finally obtained. The concentration of the fluorescent protein attached to the second protein was measured using a fluormeter. As a result, the concentrations of the three second proteins used were measured in the range of approximately 400 to 1000 nM.

실시예 3: 기판의 준비Example 3: Preparation of substrate

The coverslip was washed well with tertiary distilled water, and then washed with piranha solution (sulfuric acid: hydrogen peroxide = 2: 1 to 3: 1 (v / v)). On the surface of the washed coverslip, a coating was performed by sequentially treating a PEG mixture of aminopropylsilane, polyethylene glycol (PEG) and biotin-PEG in a ratio of 100: 3. The biotin-PEG ratio may be 100: 3 or more.

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

In the following test, the prepared coverslip and acrylic substrate were used. Before the test, the coverslip and acrylic substrate were thawed or assembled, or pre-assembled after the PEG coating was finished, and stored in -20 ℃ in assembled form. Used before thawing.

실시예 4: 제1 단백질과 제2 단백질 간 단백질-단백질 상호 작용 이미징Example 4: Protein-protein interaction imaging between first protein and second protein

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

Antibodies against the target first protein were added to the prepared substrate. At this time, the antibody used was prepared as 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 that is not conjugated to Biotin is used, the antibody of the first protein can be attached using a secondary antibody.

The antibodies against the first protein used at this time are summarized in Table 3 below:

The antibody-treated substrate was washed using the method used for the previous neutravidin washing. Prior to the prepared substrate, a tissue lysate containing the first protein prepared in Example 1 was introduced. In the case of an antigen-antibody reaction, it may continue to increase up to 15 minutes, and the antigen-antibody reaction efficiency may be lowered over time while exceeding 15 minutes, so the reaction time is set to about 15 minutes.

After the reaction, the substrate is washed in the same manner as in the previous washing method.

Thereafter, the second protein lysate obtained in Example 2 was introduced into the substrate. The concentration of the second protein in the used first protein lysate was a value between 1-50 nM (about 30 nM) based on the fluorescent protein. When the second protein concentration is 100 nM or more, background noise in the fluorescence microscope becomes large, which interferes with measuring an accurate fluorescence signal.

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

실시예 5: 단백질 복합체 (PPI complex) 분석Example 5: Protein complex (PPI complex) analysis

The protein complex was analyzed based on the toolkit provided by the Matlab program (provided by MathWorks).

The fluorescence image obtained in Example 4 was stored in 16-bit unsigned integer format. The fluorescent signal was obtained from enhanced green fluorescent protein (eGFP), 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 eGFP emission time for about 11 seconds. Among the entire frames (10 frames), the first frame was discarded, and an image of 3 frames (7-9th frame) was averaged to generate one image. This process was repeated by moving the position in the well and performing a total of 10 The following procedure was performed by acquiring images of dogs. The reason for discarding the initial 6 frames is to use the screen in which the unnecessary signal (autofluorescence) generated on the surface of the blank substrate disappears and the eGFP signal is maintained. The screening section may vary according to imaging conditions / equipment construction conditions. In this embodiment, an EMCCD (Electron-multiplying charge-coupled device; Andor iXon Ultra 897 EX2 (DU-897U-CS0-EXF)) camera is used to set the exposure time per frame to 0.1 seconds and the EMCCD gain value to 40. Fluorescence images were obtained.

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

(a) The initial start starts at the top left. One frame consists of 512x512 pixels. Based on the reference pixel, the median value was obtained from 11x11 pixels 11 to the right and 11 to the bottom, and the median value was subtracted from the value of the reference pixel [(Intensity_pixel)-(medianIntensity_11x11neighborhood)]. The above procedure was performed for all 512x512 pixels. Pepper & salt noise was removed through median filtering.

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

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

The signal from the fluorescent protein occurs in a form (localized point spread function (PSF)) aggregated at a specific location, and the number of PPI complexes between the first protein and the second protein to measure the number (physical value) of this PSF (biological Phosphorus value). The PSF value was converted to the biological value of the PPI complex through the following process:

(a) Local maximum position was obtained (eg, i-th row, j-th column pixel). As described above, the single molecule fluorescent signal is formed by gathering at a specific position (approximately 5x5 pixel size under current observation equipment, 1 pixel = 0.167 micrometer) among 512x512 pixels, so that individual PSFs can be selected by finding a local maximum. This can be obtained using the toolkit provided by Matlab.

(b) A determination is made as to whether the local maximum obtained in (a) above is actually generated from PSF. First, a minimum intensity value of a local maximum was defined, and only the case where the local maximum was greater than the minimum value 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 the 5x5 pixel information was retrieved 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 center of brightness deviates by more than 0.5 pixels from the existing local maximum coordinates (when 2D symmetry for the PSF shape disappears), it is judged as an abnormal fluorescent signal and excluded from the analysis.

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

After performing the above process for all files photographed under the same conditions, the number of PSFs was collected, and then the average and standard deviation were obtained. This value finally becomes a value indicating the number of PPI complexes under a specific condition (see FIG. 1).

실시예 6. PPI와 유방암 재발 여부와의 상관관계Example 6. Correlation between PPI and breast cancer recurrence

EGFR, HER2, and HER3, which are known to be overexpressed in breast cancer, were selected as the first protein, and PLCG1 (PLC-gamma1), PI3K, and Grb2, their representative sub-signaling pathway proteins, were selected as the second protein. 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 value of the PPI complex in the case where only the second protein was treated without sample processing on the substrate prepared in Example 3 was used. The subtracted value (PPI count) was determined:

PPI count = [PPI complex value measured in sample]-[PPI complex value measured when sample is not processed]

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

(In the above table, a sample marked with 'O' for recurrence means a relapsed sample)

As shown in Table 4 and FIGS. 2A to 2B (dark bars indicate recurrent patients), the PPI count is 0 or less (ie, the PPI value in the sample is the noise value of the measurement system or the background signal value (ie, the sample is not processed). HER2-PLCG1, which has the highest percentage of non-recurring patients among samples), which is lower than the measured PPI complex value), has the highest percentage of relapsed patients among samples whose PPI count is greater than 0, so HER2-PLCG1 PPI count Was selected as a biomarker for breast cancer recurrence.

실시예 7. HER2-PLCG1 PPI count>0인 경우의 HER2 또는 HER3 발현량과 유방암 재발 여부와의 상관관계Example 7. Correlation between HER2 or HER3 expression and breast cancer recurrence when HER2-PLCG1 PPI count> 0

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

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

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

After completion of the reaction, the number of single-molecule PSFs is measured in the same way as the PPI complex measurement through total reflection microscopy to quantify the amount of each first protein.

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

As can be seen in Figure 3a, among the 14 samples with a higher HER2 expression level than the average value of the HER2 expression level of the samples with HER2-PLCG1 PPI count> 0, 8 samples had recurrence of breast cancer and 6 samples did not relapse. Of the 33 samples with HER2 expression level lower than the mean value, 3 of the breast cancer recurred samples and 30 non-recurred samples showed the percentage of breast cancer recurrence in samples with a higher HER2 expression level than the average value ([ 8/14] * 100 = 57%) was found to be significantly higher than the rate of breast cancer recurrence ([3/33] * 100 = 9%) in samples with HER2 expression levels below the mean value.

These results, after measuring the HER2-PLCG1 PPI count, when the HER2 expression level was measured for a sample having HER2-PLCG1 PPI count> 0 in succession, when the HER2 expression level was higher than the average value of the samples with HER2-PLCG1 PPI count> 0 It is a result confirming 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:

Reliability of patient recurrence judgment (Positive Prediction Value, PPV;%) = [(Number of relapsed samples among samples with a biomarker expression level exceeding an average value or an average value + SD (standard deviation)) / (average or average value of biomarker expression levels + Total number of samples exceeding SD)] * 100;

Non-recurring patient judgment reliability (Negative Prediction Value, NPV;%) = [(Number of non-recurring samples among samples with a biomarker expression level below the mean value or mean value + SD) / (() of samples with a biomarker expression level below the mean value or mean value + SD Total number)] * 100;

Decision rate of recurring patients (Sensitivity;%) = [(Number of recurring samples among samples whose biomarker expression level is above the mean value or the mean value + SD) / (total number of recurring samples)] * 100;

Exclusion rate of non-recurring patients (Specificity;%) = [(Number of non-recurring samples among samples whose biomarker expression level is below the mean value or the mean value + SD) / (total number of non-recurring samples)] * 100;

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

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

As shown in Table 6 and Table 7, when the HER2-PLCG1 PPI count> 0 samples are evaluated by measuring the expression level of HER2 or HER3 as a biomarker, the results are generally excellent in breast cancer recurrence diagnosis indicators. In particular, when the expression level of HER2 was measured and evaluated for samples with HER2-PLCG1 PPI count> 0, the reliability of recurrent patient determination, the reliability of non-recurring patient judgment, the rate of recurring patient determination, and the rate of exclusion of non-recurring patients Better results were obtained in all evaluated indicators of.

실시예 8.Example 8. 바이오마커 발현 수준 측정 후 HER2-PLCG1 PPI 측정하여 유방암 재발과의 상관관계 확인Measurement of biomarker expression level and measurement of HER2-PLCG1 PPI to confirm correlation with breast cancer recurrence

The correlation between breast cancer recurrence and HER2-PLCG1 PPI count was measured and biomarker (HER2 or HER3) expression level was measured.

More specifically, for all of the samples prepared in Example 1 (73), the expression levels of HER2 and HER3 as biomarkers were measured, respectively, with reference to the method of Example 7, and the expression levels were higher than the average value. Was selected. Then, with respect to the selected sample, HER2-PLCG1 PPI count was measured with reference to the method of Example 6, and a sample with HER2-PLCG1 PPI count> 0 was selected to determine patient determination reliability, non-recurring patient determination reliability, and recurrence patient determination. Indicators, and indicators of non-relapse patient exclusion rate were measured.

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

실시예 9. 진단 성능 평가Example 9. Assessment of diagnostic performance

The results of the results obtained in Examples 6 to 8 were evaluated for breast cancer recurrence diagnosis performance through positive / negative ratios (diagnostic ratios) and Youden's index, and the results are shown in Table 10 below:

(LR +, Possibility ratio: [Recurrence sample rate among recurrence positive (+) judged samples / Recurrence sample rate among recurrence negative (-) judged samples], the higher the value, the better the predictive performance,

LR-, Negative Probability Ratio: [Ratio of actual non-recurring samples among samples with recurrence positive (+) / ratio of actual non-recurring samples among samples with recurrence negative (-)], the smaller the value, the better the predictive performance,

Youden's index: Sensitivity + Specificity-1; If the Sensitivity and Specificity are expressed in%, the 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 Table 8 are analyzed by ROC curve analysis are shown in Table 11 and FIG. 6 (ROC curve) below.

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

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

Area is the integral value of the area under the graph, standard error is the error due to the inaccuracy that occurs when connecting the points that make up the graph, and Asymptotic Probability (p-value) is a recurring patient when the sample is accidentally selected. As an indicator of how far from the probability of being found, the smaller the number, the lower the probability, which means that the diagnostic accuracy is higher.

UCL and LCL represent a range of Area values when assuming 95% reliability, which means that the probability that an accurate Area value is between LCL <Area <UCL is 95%.

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

As shown in Tables 10, 11, and 6 above, the method of evaluating by performing HER2-PLCG1 PPI count measurement and HER2 or HER3 expression measurement is the best in both the tested indicators, in particular, both Sensitivity and Specificity. You can get results,

As a result of calculating the quantitative diagnostic performance indicators Youden's index (Sensitivity + Specificity-1) and the positive / negative probability ratio (LR + / LR-), it was also shown that it was excellent in predicting whether breast cancer recurred.

In particular, the HER2-PLCG PPI count measurement and the HER2 expression level measurement were performed regardless of the order, and the Youden's index in the method was evaluated to exceed 0.5 and showed the closest to 1, and these results predicted whether the method would recur breast cancer. It means that it shows very good performance.

When synthesizing the above results, the method of evaluating by performing HER2-PLCG PPI count measurement and HER2 or HER3 expression level, in particular, HER2 expression level measurement provided in this specification is a recurrence patient determination reliability, non-recurrence patient determination reliability, recurrence patient It can be said that it shows excellent results in all indicators of the determination rate and the non-relapse patient exclusion rate, and also shows excellent breast cancer recurrence prediction performance in the results evaluated by the ratio of positive / negative and Youden's index.

실시예 10: 유방암 환자로부터 수득한 시료의 HER2 발현수준 측정Example 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 mixture of polyethylene glycol (PEG) and biotin-PEG containing PEG. After washing the substrate, an antibody against the HER2 protein was treated. At this time, the antibody used was prepared as 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 that is not conjugated with biotin, 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 introduced. In the case of an antigen-antibody reaction, it may continue to increase up to 15 minutes, and the antigen-antibody reaction efficiency may be lowered over time while exceeding 15 minutes, so the reaction time is set to about 15 minutes.

Thereafter, a HER2 antibody to which a fluorescent protein was attached was introduced. The HER2 antibody used at this time is characterized by acting on a different site from the antibody attached to the substrate. The substrate was fixed and imaged on a fluorescence microscope, and the fluorescence signal of the fluorescent protein was measured to obtain data. In order to improve the accuracy of the results, images were acquired at a total of 10 different locations under the same conditions, and averaged and used as representative values.

실시예 11: 기준 값과 비교하여 유방암 재발여부 예측Example 11: Prediction of breast cancer recurrence compared to a reference value

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

As the number of samples of the patient (i.e., the N value) increases, the reference value may vary somewhat as data is accumulated, but the accuracy may be higher. When the signal value measured in Example 10 was greater than or equal to the reference value, it was predicted that the prognosis for breast cancer was bad, and if it was lower than the reference value, the prognosis was good.

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

FIG. 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 assay using the HER2 level measurement method provided herein, and the HER2 by the IHC assay Even patients classified as (-) (HER2 0 or 1) show a large difference in HER2 levels when measured by the HER2 level measurement method provided herein, and among patients classified as HER2 (-), patients with significant levels of HER2 expression It shows that it can exist.

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

실시예 12: 유방암 재발여부 예측과 실제 생존율의 상관관계 분석Example 12: Analysis of correlation between breast cancer recurrence prediction and actual survival rate

For the 72 samples used for setting the reference value in Example 11, the measurement signal value of the sample was divided into a high risk group higher than a reference value and a low risk group lower than the reference value. The high risk group was 9 and the row risk group was 63. FIG. 69 shows the correlation between prediction of breast cancer recurrence and actual survival rate (Disease Free Survival) by reflecting the results of 72 months of follow-up surveys of 72 breast cancer patients who acquired the sample. 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 confirmed that the survival rate of the high-risk group is significantly lower than the low-risk group, thereby validating the recurrence diagnosis technique performed in this embodiment.

실시예 13: HER2 표적치료요법 적용 환자군 선정단계Example 13: HER2 target therapy application patient group selection step

In Examples 10 and 11, the high-risk group above the reference value has a relatively high expression level of HER2, and can be selected as a patient group effective for HER2 target treatment. The HER2 target treatment is a drug targeting the HER2 protein, and typically includes Herceptin (trastuzumab), and other HER2-TKI or pan-HER TKI, but is not limited thereto. It can be used as a companion diagnostic method of HER2 target therapy because it can be classified as HER2 (-) and target group can receive HER2 target therapy.

실시예 14: 유방암 샘플의 확장 및 유방암 재발 여부 예측Example 14: Expansion of breast cancer samples and prediction of breast cancer recurrence

In a breast cancer tissue sample of 72 breast cancer patients used in Example 11, ER (estrogen receptor) / PR (progesterone receptor) positive (

steps

2 or 3 in IHC test) and HER2 negative (

step

0 or 1 in HER2 IHC test) ( A total of 138 breast cancer tissue samples were prepared by adding a breast cancer tissue sample of 66 breast cancer patients determined as ER / PR +, HER2-).

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

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

The results of analyzing the HER2 expression level of the obtained 138 breast cancer samples, their classification results into high risk and low risk, and recurrence rates are shown in FIG. 10. In addition, by reflecting the results of a follow-up survey of 138 breast cancer patients who obtained the sample for 120 months, the correlation between prediction of breast cancer recurrence and actual survival (Disease Free Survival) was measured and shown in FIG. 11. As shown in FIG. 10, breast cancer recurred in 28 patients out 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 9 patients, whereas the recurrence rate was 29% in 9 of the 31 samples that were classified as high-risk group. It was found to be about twice as high, which shows that the p-value by log-rank test is statistically significant at about 0.014, making it possible to predict high risk of recurrence. In addition, as shown in FIG. 11, it can be confirmed that the survival rate of the high-risk group (dashed line) is significantly reduced at a significantly faster rate compared to the low-risk group (solid line), thereby demonstrating the effectiveness of the recurrence diagnosis technique performed in this embodiment. Can be.

Claims

(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 on predicting breast cancer prognosis, comprising:
According to claim 1,

Proceed to the steps (I) and (II) above,

After step (I),

(I-a) further comprising comparing the protein-protein interaction level between 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 of the biological sample measured in step (I) is higher than 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 without adding a biological sample or a biological sample and PLC-gamma protein.

How to provide information to predict breast cancer prognosis.
According to claim 2,

After step (II), further comprising (II-a) comparing the expression level of HER2 or HER3 measured in step (II) with a reference expression level,

How to provide information to predict breast cancer prognosis.
According to claim 1,

Proceed to the above 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 a reference expression level as a result of the comparison of step (II-a), the protein between HER2 and PLC-gamma protein of the biological sample -Performed by measuring protein interactions,

The reference expression level is an average value of the expression level of HER2 or HER3 in a sample isolated from two or more breast cancer patients, with or without the biological sample being separated,

How to provide information to predict breast cancer prognosis.
According to claim 4,

After step (I),

(I-a) further comprising comparing the reference protein-protein interaction level to a protein-protein interaction level between HER2 and PLC-gamma protein of the biological sample measured in step (I),

How to provide information to predict breast cancer prognosis.
According to any one of claims 1 to 5, wherein the breast cancer,

(1) HER2 negative (-) breast cancer,

(2) estrogen receptor (ER), progesterone receptor (PR), or both are positive (+) breast cancer, or

(3) breast cancer having the characteristics of (1) and (2) above,

How to provide information to predict breast cancer prognosis.
The method of claim 6,

The HER2 negative (-) breast cancer has a score (IHC score) of 1 or less determined by immunohistochemical staining (IHC) using a HER2 specific antibody,

The estrogen receptor (ER), progesterone receptor (PR), or both of these positive (+) breast cancer scores are determined by immunohistochemical staining (IHC) using ER specific antibodies, PR specific antibodies or both. (IHC score) is 2 or more,

How to provide information to predict breast cancer prognosis.
The method according to any one of claims 1 to 5, wherein the breast cancer prognosis is breast cancer recurrence.
(i) measuring the HER2 protein expression level of a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) with a reference expression level

Including,

The reference expression level is an average value or an average value ± standard deviation (SD) of the expression level of HER2 protein in a sample separated from two or more breast cancer patients with or without the patient with the biological sample isolated,

The breast cancer,

(1) HER2 negative (-) breast cancer,

(2) estrogen receptor (ER), progesterone receptor (PR), or both are positive (+) breast cancer, or

(3) breast cancer having the characteristics of (1) and (2) above,

How to provide information to predict breast cancer prognosis.
The method of claim 9,

The HER2 negative (-) breast cancer has a score (IHC score) of 1 or less determined by immunohistochemical staining (IHC) using a HER2 specific antibody,

The estrogen receptor (ER), progesterone receptor (PR), or both of these positive (+) breast cancer scores are determined by immunohistochemical staining (IHC) using ER specific antibodies, PR specific antibodies or both. (IHC score) is 2 or more,

How to provide information to predict breast cancer prognosis.
The method of claim 9 or 10, wherein the breast cancer prognosis is breast cancer recurrence.
(i) measuring the HER2 protein expression level of a biological sample isolated from a breast cancer patient, and

(ii) comparing the HER2 protein expression level measured in step (i) with a reference expression level

Including,

The reference expression level is an average value or an average value ± standard deviation (SD) of the expression level of HER2 protein in a sample separated from two or more breast cancer patients with or without the patient with the biological sample isolated,

The breast cancer,

(1) HER2 negative (-) breast cancer,

(2) estrogen receptor (ER), progesterone receptor (PR), or both are positive (+) breast cancer, or

(3) breast cancer having the characteristics of (1) and (2) above,

A method to provide information on screening patients suitable for HER2 targeted treatment.
The method of claim 12, after step (ii),

(iii) further comprising selecting a breast cancer patient from which a biological sample having a HER2 protein expression level measured in step (i) above a reference expression level is suitable for HER2 target treatment,

A method to provide information on screening patients suitable for HER2 targeted treatment.