WO2019098509A1 - Biomarker for diagnosis of breast cancer and use thereof - Google Patents

Abstract

Provided according to an embodiment are a composition and a kit for diagnosis of breast cancer and a method for the diagnosis of breast cancer or for the provision of information for breast cancer diagnosis, using same. Thus, breast cancer can be diagnosed early. In addition, mammary cancer can be diagnosed in companion animals, whereby the likelihood of onset of breast cancer in persons who raise the companion animals can be predicted.

Description

Biomarkers for diagnosis of breast cancer and uses thereof

A kit for diagnosing breast cancer or breast cancer, a diagnostic method of breast cancer using the kit, or a method of providing information for diagnosing breast cancer.

Human breast or breast cancer is the second most common malignancy of female cancer. The causes of breast cancer vary, but only 20% of known causes are present. About 80% of the remaining cases are unknown, but there is a strong opinion that it is likely to be caused by an external environment. Diseases caused by the external environment are too numerous to explain because of the causes. Therefore, a complex external environment is considered to be the main cause of breast cancer. Diseases caused by external environmental causes can not be predicted and prevented in advance, and since the treatment process is performed only after the diagnosis of disease is made, there is a problem that it takes considerable time for cost, period and recovery.

Companion animals, such as companion dogs, are exposed to the same space and environment as humans and receive a similar external environment to humans. Dogs are more metabolizable than humans and have a higher susceptibility to disease in the external environment, so they may be able to first express certain diseases that may occur in a person (eg, a guardian of a dog). For example, the development of breast cancer in a dog can mean the possibility of breast cancer in humans.

Therefore, it is necessary to develop a method for predicting the onset of human breast cancer caused by the environment by first diagnosing the breast cancer of a dog who lives in a human-like environment.

There is provided a composition for diagnosing breast cancer,

A kit for diagnosing breast cancer is provided.

A diagnostic method for breast cancer or a method for providing information for diagnosing breast cancer.

One aspect provides a composition for breast cancer diagnosis comprising an agent that measures the expression level of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A.

The ZG16B may also be referred to as a zymogen granule protein 16 homolog B protein. The ZG16B is a uniprot. Lt; RTI ID = 0.0 > Q96DA0. ≪ / RTI > In the case of ZG16B, the number of Uniprot No. is ZG16B. Lt; RTI ID = 0.0 > F1Q2Y5. ≪ / RTI >

The CCL23 is a chemokine (CC motif) ligand 23 protein, a macrophage inflammatory protein 3 (MIP-3), and a myeloid progenitor inhibitory factor 1 (MPIF) -1). The CCL23 is mainly expressed in lung and liver tissues and can be expressed in bone marrow and placenta. The CCL 23 is a Uniprot. Lt; RTI ID = 0.0 > P55773. ≪ / RTI > In the case of the CCL 23, there are four Uniprot No.s. Lt; RTI ID = 0.0 > J9PBF2. ≪ / RTI >

The ALP may also be referred to as an alkaline phosphatase protein or a basic phosphatase. The ALP may be a protein located in the extracellular exosome. The ALP is a Uniprot No. in human. 0.0 > P05186. ≪ / RTI > In the case of the ALP, there are four Uniprot No.s. Lt; RTI ID = 0.0 > F1PF95. ≪ / RTI >

ITGB2 may also be referred to as integrin beta-2 or CD18 protein. The ITGB2 may be a protein located in the extracellular exosome. The ITGB2 is a Uniprot No. for human. Lt; RTI ID = 0.0 > P05107. ≪ / RTI > The ITGB2 is Uniprot No. in case of dog. Lt; RTI ID = 0.0 > F1Q1H6 < / RTI > or Q9TU04.

The CLIC6 may also be referred to as Chloride intracellular channel protein 6 or CLIC1L protein. The CLIC6 may be a protein located in the extracellular exosome. In the case of a human, the CLIC6 is a Uniprot No.. Lt; RTI ID = 0.0 > Q96NY7. ≪ / RTI > In the case of the CLIC6, there are four Uniprot No.s. Lt; RTI ID = 0.0 > F1P740. ≪ / RTI >

The ATP6V1B1 may also be referred to as a V-type proton ATPase B, a V-type proton ATPase subunit B, or a kidney isoform protein. The ATP6V1B1 may be a protein located in the extracellular exosome. In the case of human, ATP6V1B1 is expressed by Uniprot No. 1. Lt; RTI ID = 0.0 > P15313. ≪ / RTI > In the case of ATP6V1B1, Uniprot No. < / RTI > E2R1W5 or J9NUJ1. ≪ / RTI >

The FAM162A may also be referred to as a human growth and transformation-dependent protein (HGTD-P) or an E2-induced gene 5 protein (E2IG5). The FAM162A may be a protein located in the extracellular exosome. In the case of human, FAM162A is a Uniprot. Or an amino acid sequence represented by Q96A26. In the case of FAM162A, the FAM162A has Uniprot No.. E2QRU5. ≪ / RTI >

The fragment may be an immunogenic polypeptide as part of the protein.

The term " expression level " means the amount of the protein or the amount of the transcript. The expression level may be a relative proportion of the protein or transcript. For example, an increase in expression level may be an increase in the amount of protein or transcript compared to negative control.

The agent may be an antibody that specifically binds to the protein or a fragment thereof, or an antigen-binding fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. The term " antibody " can be used interchangeably with the term " immunoglobulin ". The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen binding fragment refers to a polypeptide comprising an antigen binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a surface of a metal chip, plate, or well.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The nucleic acid may be a primer or a probe. The primer or the probe may be labeled at the end or inside thereof with a fluorescent substance, a chemiluminescent substance or a radioactive isotope. Wherein the primer has at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50% of the nucleotide sequence of SEQ ID NOS: Or < RTI ID = 0.0 > identical. ≪ / RTI >

The term " breast cancer " refers to breast cancer, and may also be referred to interchangeably as " mammary tumor ". The breast cancer may include mammary gland breast cancer, lobule breast cancer, or a combination thereof. Breast cancer can be classified into two types depending on the site of development: cancers arising in the ductal and lobular epithelium and cancers arising from the substrate. The breast cancer may include a complex carcinoma (CC) or a ductal carcinoma (DC). Tuberculous carcinoma is a type of breast cancer that exists principally in the ducts of individuals.

The term " diagnosis " refers to the determination of a disease name, and may include a disease name of a breast cancer, a disease state, stage, etiology, presence or absence of complication, prognosis, and recurrence.

The composition may be for simultaneous or sequential diagnosis of dog mammary tumors and human breast cancer. For example, the composition can be used to diagnose human breast cancer after it has been used to diagnose breast cancer.

Another aspect provides a kit for breast cancer diagnosis comprising an agent that measures the expression level of a protein selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A or a fragment thereof.

An agent for measuring the expression level of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, FAM162A, fragment, protein or fragment thereof, breast cancer, and diagnosis are as described above.

The kit may further comprise a sample necessary for measuring the expression level of the protein or fragment thereof. The kit may comprise a substrate, a suitable buffer solution, a chromogenic enzyme, a secondary antibody labeled with a fluorescent substance, or a chromogenic substrate for immunological detection of a solid support, antibody or antigen-binding fragment. The kit may comprise a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent material, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Another aspect is a method of detecting the expression level of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A in a biological sample isolated from a subject suspected of having breast cancer. And

And comparing the measured expression level to an expression level of a protein or fragment thereof of a normal control.

An agent for measuring the expression level of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, FAM162A, fragment, protein or fragment thereof, breast cancer, and diagnosis are as described above.

The method comprises measuring the expression level of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A in a biological sample isolated from a subject suspected of having breast cancer.

The subject may be a mammal such as a human, dog, cat, mouse, rabbit, horse, sheep, hamster, hedgehog, ferret, or guinea pig. The subject may be an individual suspected of having or afflicted with breast cancer.

The biological sample refers to a sample obtained from the individual. The biological sample may be a blood sample, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural effusion, aspiration nipple, lymphatic fluid, airway fluid, intestinal fluid, urinary reproductive fluid, breast milk, Body fluids, ascites, cystic tumor body fluids, saline fluids, or a combination thereof.

Wherein the measuring step comprises incubating the antibody or antigen-binding fragment thereof that specifically binds to the protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A .

The measurement may be performed by any of a variety of methods including electrophoresis, immunoblotting, enzyme linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, mass spectrometry, microarray, Northern blotting, A polymerase chain reaction (PCR), or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real time PCR or reverse transcription PCR.

The method comprises comparing the measured level to an expression level of a protein or fragment thereof of a normal control.

The term " normal control " can be used interchangeably with the term " negative control ". The normal control may be an individual that has never been breast cancer or a healthy individual.

If the measured expression level of the protein or fragment thereof is increased relative to the expression level measured in a normal control, the subject may further include a step of determining that the subject is at risk of developing breast cancer or having breast cancer.

Another aspect is a method of detecting the expression level of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A in a biological sample isolated from a subject suspected of having breast cancer. And

And comparing the measured expression level to an expression level of a protein or fragment thereof of a normal control, to provide information for diagnosing breast cancer.

The method may further include predicting the probability of developing breast cancer of the second individual through the diagnosis of breast cancer of the individual. The entity (i.e., the first entity) may be a companion animal and the second entity may be a person. The companion animal may refer to an animal living in the same living environment as a person. The companion animal may be a dog, a cat, a mouse, a rabbit, a hamster, a hedgehog, a pereote, or a guinea pig. Because one of the major causes of breast cancer is environmental factors, breast cancer can be diagnosed in companion animals living in a human environment to provide early diagnosis of breast cancer in a person who is the protector of a companion animal or information necessary for breast cancer diagnosis have. For example, diagnosis of breast cancer in a dog can predict the likelihood of breast cancer in a person who is a guardian of a dog.

According to a composition for breast cancer diagnosis according to one aspect, a kit, and a method for diagnosing breast cancer using the kit, or a method for providing information for diagnosing breast cancer, the breast cancer can be diagnosed at an early stage. It is also possible to diagnose mammary carcinoma in companion animals and to predict the likelihood of developing breast cancer in a person who is a protector of companion animals.

Fig. 1 shows the analysis of proteins differentially expressed in mammary tumor tissues and nearby normal tissues (p-value? 0.05, fold change? 2).

FIG. 2a shows a Venn diagram analysis of mammary tumor tissues 17T, 23T, and 26T, and a list of proteins present in the extracellular exosome among the proteins up-regulated only in tubular carcinoma 17, FIG. 2B shows a list of proteins present in mammary tissues 9T, 18T, Diagram analysis and lists of proteins present in the extracellular space or in the extracellular space among the proteins up-regulated only in mixed carcinoma 9T.

FIG. 3 is a graph showing expression levels in normal tissues and tumor tissues of the selected biomarkers.

FIG. 4A is a graph showing the amount (pg / ml) of blood CCL23 in the normal group and the breast cancer group, and FIG. 4B is an ROC curve showing the specificity and sensitivity of CCL23 for breast cancer diagnosis.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

Example 1. Identification of Biomarkers in Blood of Patients with Breast Cancer

1. Identification of differentially expressed proteins in mammary gland tissue of dogs

Proteins differentially expressed in mammary tumor tissues were isolated from dogs with mammary gland carcinoma.

Specifically, 10 dogs were isolated from female dogs with mammary gland carcinoma (Supplied by Seoul National University Animal Hospital). Tissue (Promega) was used to dissociate the isolated tissue into peptides. The digested peptide samples were purified using a C ₁₈ column (Thermo Fisher Scientific Inc.). The purified peptides were subjected to mass spectrometry (Thermo Fisher Scientific Inc.) to produce raw data according to the mass of the peptides. Using the raw data, we analyzed the proteins that were differentially expressed in the mammary gland tissues of the dogs and nearby normal tissues. Volcano plots were used to analyze differences in expression according to statistical significance.

Figure 1 shows p-value ≤0.05, fold change ≥2, data of protein differentially expressed in breast cancer tissue and nearby normal tissue. In Fig. 1, the numbers indicate the number of dogs, " T " means breast cancer tissue, and " N " means normal tissue. For example, "9T vs 9N" refers to normal tissue of 9 dogs versus 9 dogs. In addition, the red dot indicates an increased protein in mammary tissue when compared to normal tissue, and the blue dot indicates a decreased protein in mammary tissue when compared to normal tissue.

As shown in Fig. 1, proteins differentially expressed in mammary carcinoma were identified as compared with normal tissues.

2. Identification of differentially expressed proteins according to cancer type

We analyzed proteins that differed statistically from ductal carcinoma or mixed carcinoma compared with normal normal tissue.

The nearby normal tissues and cancer tissues were obtained from the dog colleagues in Seoul National University Animal Hospital and Haemaru Animal Hospital. Tumor tissues and corresponding normal tissues were treated within 5 minutes of tissue isolation in vivo. The isolated tissue was divided into pea-sized segments and stored in liquid nitrogen.

The carcinoma of the specimen was confirmed and confirmed as ductal carcinoma, ductal adenoma, mixed carcinoma, mixed adenoma and adenoma.

carcinoma	Name of sample
Tuberous carcinoma	17T
Tuberous adenoma	23T, 26T
Mixed carcinoma	9T
Mixed type adenoma	18T, 19T
Adenoma	22T, 24T, 49T, 50T

As described in 1., a Venn diagram was used to identify proteins whose expression increased only in carcinoma in a comparative analysis of dogs. In order to confirm the intracellular location of proteins expressed only in carcinoma, gene ontology was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery, https://david.ncifcrf.gov/home.jsp) Analysis was performed. In the results of the gene presence analysis, extracellular exosome and extracellular space proteins released into the extracellular space of the analyzed proteins were selected to enable diagnosis in body fluid such as blood. Based on the analysis of the diagram and the analysis of the presence of genes, proteins expressed up-regulated in the mammary carcinoma and present in the extracellular space or in the extracellular space are shown in FIGS. 2A and 2B.

Figure 2a shows a Venn diagram analysis of mammary tumor tissues 17T, 23T, and 26T and a list of proteins present in extracellular exosomes among 326 proteins up-regulated only in tuberous carcinoma 17T.

FIG. 2B shows a Venn diagram analysis of mammary tumor tissues 9T, 18T, and 19T and a list of proteins present in the extracellular space or extracellular space among 276 proteins up-regulated only in mixed carcinoma 9T.

3. Selection of biomarkers that are differentially expressed in tubular carcinomas

Based on the results of the analysis described in Section 2, we analyzed proteins that differ significantly in ductal carcinoma compared to nearby normal tissues.

Expression levels in normal tissues were calculated by calculating expression levels in tubular carcinomas. Proteins and expression ratios that are significantly differentially expressed in coronary carcinomas are shown in Table 2 below.

Of the 326 proteins whose expression was significantly increased only in ductal carcinoma of the breast, proteins with 10 times or more difference in expression compared to normal tissues were selected. Among the selected biomarkers, biomarkers reported to be related to breast cancer were excluded from the existing articles.

Finally, new biomarkers ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A were selected which showed a significant increase in expression in ductal carcinoma carcinomas compared with normal tissues.

4. Screening of differentially expressed biomarkers in mixed carcinomas

Based on the results of the analysis described in Section 2, we analyzed proteins that were statistically significantly differentially expressed in mixed carcinomas compared to nearby normal tissues.

Expression levels in the mixed tissues of normal tissues were calculated to calculate expression levels. Proteins and expression levels that are significantly differentially expressed in mixed carcinomas are shown in Table 3 below.

Of the 276 proteins whose expression was significantly increased only in mixed carcinomas of mammary carcinomas, proteins with 10 times or more difference in expression compared to normal tissues were selected. Among the selected biomarkers, biomarkers reported to be related to breast cancer were excluded from the existing articles.

The new biomarkers ZG16B, CCL23, and CHID1, which showed a significant increase in expression in mixed carcinomas compared to normal tissues, were identified and ZG16B and CCL23, which have high differential expression ratios, were selected.

Myoepithelial carcinomas or mixed carcinomas are rare in humans, making it difficult to find and study biomarkers. Therefore, the biomarkers ZG16B and CCL23 identified in dogs were selected as breast cancer prediction markers applicable to human as well as dogs.

5. Verification of biomarkers

Six pairs of mammary tumor tissues and normal tissues (individual numbers # 1, # 11, # 72, # 125, # 127, and # 132) were prepared as described in step 1 and total RNA was extracted from the prepared tissues.

Real-time polymerase chain reaction (qRT-PCR) was performed for total RNA. The reaction conditions of qRT-PCR were incubation at 95 ° C for 5 minutes, followed by 40 cycles of 95 ° C for 15 seconds, 60 ° C for 15 seconds, and 72 ° C for 25 seconds as one cycle. Thereafter, incubation was performed at 72 캜 for 5 minutes, at 65 캜 for 0.5 seconds, at 95 캜 for 5 seconds, and at 12 캜 for 20 minutes. The primers used are shown in Table 4 below.

Target gene	primer	Nucleotide sequence
CCL23	Forward	5'-CCCAATCTCACCCCACGAAA-3 '(SEQ ID NO: 1)
	Reverse	5'-AGGGGTTAGCACAGACAAGC-3 '(SEQ ID NO: 2)
CHID1	Forward	5'-GGGCTTACGTATAGTGCCCC-3 '(SEQ ID NO: 3)
	Reverse	5'-CGAAGCCGTCAAAGTGTTGG-3 '(SEQ ID NO: 4)
ZG16B	Forward	5'-ATGTACGGGATTGGGCAAGG-3 '(SEQ ID NO: 5)
	Reverse	5'-AGTGATGTACTCTCCCGGCT-3 '(SEQ ID NO: 6)
CLIC6	Forward	5'-TTCTCCCAGCGCCTTTTCAT-3 '(SEQ ID NO: 7)
	Reverse	5'-TCCGTCTTGACTTCGCCATC-3 '(SEQ ID NO: 8)
ATP6V1B1	Forward	5'-CTGACAGCCCAGATTGGGAA-3 '(SEQ ID NO: 9)
	Reverse	5'-AGACACCTCAAGCACCTGTC-3 '(SEQ ID NO: 10)
FAM162A	Forward	5'-ACTTACAGCCACAGAGTGCC-3 '(SEQ ID NO: 11)
	Reverse	5'-AGATGCATCCTGCCACTGTC-3 '(SEQ ID NO: 12)
ALP	Forward	5'-CCAAGGACGCTGGGAAATCT-3 '(SEQ ID NO: 13)
	Reverse	5'-ACGTTGTGCATGAGCTGGTA-3 '(SEQ ID NO: 14)
ITGB2	Forward	5'-ATCGGGAAGAACTGCGAGTG-3 '(SEQ ID NO: 15)
	Reverse	5'-GTTCTTGTTGGGAACGTCGC-3 '(SEQ ID NO: 16)

qRT-PCR results were analyzed, normalized to the amplification results of A5B, and relative mRNA expression levels were calculated. Relative mRNA expression levels in normal and breast cancer tissues are shown in Figure 3. As shown in Figure 3, the selected biomarkers were expressed higher in mammary tumor tissue than in normal. In particular, the expression of CCL23, CLIC6, ALP, and ITGB2 was higher than normal in over 50% of the measured breast cancer tissues.

6. Verification of biomarkers using human blood

Plasma samples of normal (n = 19) and breast cancer (n = 48) plasma were collected in order to determine whether the identified biomarkers differentially expressed in dog tissue of dogs were available for human breast cancer diagnosis Provided by Samsung Medical Center.

Serum proteins were obtained from prepared plasma samples and CCL23 protein in blood was quantified using CCL23 Human ELISA kit (Invitrogen).

Based on the results of ELISA, receiver operating characteristic (ROC) curves for the diagnosis of breast cancer were generated. As a result of the ELISA, a graph of the amount of CCL23 (pg / ml) in the normal group and the breast cancer group is shown in FIG. 4A, and the ROC curve of the specificity and sensitivity is shown in FIG.

As shown in FIG. 4A, the amount of CCL23 in the blood was significantly increased in human breast cancer blood (p value <0.0001). Also, as shown in FIG. 4B, the area under curve (AUC) of the ROC curve was 0.79.

Therefore, CCL23 was confirmed to be excellent in accuracy and sensitivity as a blood biomarker for human breast cancer diagnosis.

Claims (15)

1. Wherein the expression level of the protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A is measured.
2. The composition according to claim 1,

   The substance that specifically binds to the protein or fragment thereof may be an antibody, or antigen-binding fragment thereof; or

   Wherein the polynucleotide is a nucleic acid comprising a polynucleotide that is the same as or complementary to a polynucleotide encoding the protein or fragment thereof.
3. 3. The composition of claim 2, wherein the antibody is a polyclonal antibody or a monoclonal antibody.
4. 3. The composition of claim 2, wherein the nucleic acid is a primer or a probe.
5. The composition according to claim 1, wherein the breast cancer is a complex carcinoma (CC) or a ductal carcinoma (DC).
6. The composition according to claim 1, wherein said composition is for simultaneous or sequential diagnosis of dog mammary tumors and human breast cancer.
7. Wherein the expression level of a protein selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A or a fragment thereof is measured.
8. Measuring the level of expression of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A in a biological sample isolated from a subject suspected of having breast cancer; And

   And comparing said measured expression level to expression levels of a protein or fragment thereof of a normal control.
9. 9. The method of claim 8, wherein the subject is a human, dog, cat, mouse, rabbit, horse, sheep, hamster, hedgehog, ferret, or guinea pig.
10. The method of claim 8, wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural effusion, aspiration nipple, lymphatic fluid, airway fluid, intestinal fluid, urogenital fluid, , Cerebrospinal fluid, intracranial fluid, ascites, cystic tumor fluid, positive fluid, or a combination thereof.
11. 9. The method of claim 8, wherein the step of measuring the amount of the protein or fragment thereof comprises measuring the amount of the protein or fragment thereof by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, Mass spectrometry, microarray, northern blotting, polymerase chain reaction (PCR), or a combination thereof.
12. 9. The method of claim 8, further comprising the step of determining if the measured expression level of the protein or fragment thereof is increased relative to the expression level measured in a normal control, the subject is determined to be susceptible to breast cancer or to be at risk of breast cancer / RTI &gt;
13. Measuring the level of expression of a protein or fragment thereof selected from the group consisting of ZG16B, CCL23, ALP, ITGB2, CLIC6, ATP6V1B1, and FAM162A in a biological sample isolated from a subject suspected of having breast cancer; And

    And comparing said measured expression level to expression levels of a protein or fragment thereof of a normal control.
14. 14. The method of claim 13, further comprising predicting the likelihood of developing a breast cancer of the second individual through breast cancer diagnosis of the subject.
15. 15. The method of claim 14, wherein the entity is a companion animal and the second entity is a human.

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