KR20240136713A - Composition for predicting or diagnosing bone metastasis of breast cancer and kit containing the same - Google Patents

Abstract

The composition and kit of the present invention relate to a composition and kit for predicting or diagnosing bone metastasis in breast cancer patients. The composition, kit, and method of the present invention can effectively predict and diagnose bone metastasis cancer by analyzing the level of TOR1B mRNA expression at the bone metastasis stage of breast cancer patients. In addition, the survival rate of breast cancer bone metastasis patients, etc. can be improved through the method of providing information for predicting or diagnosing bone metastasis in breast cancer patients of the present invention.

Description

COMPOSITION FOR PREDICTING OR DIAGNOSING BONE METASTASIS OF BREAST CANCER AND KIT CONTAINING THE SAME

The present invention relates to a composition for predicting or diagnosing bone metastasis of breast cancer and a kit comprising the same.

Breast cancer is the second most common cancer after lung cancer, and is the fifth most common malignant tumor in terms of mortality. The causes of breast cancer are diverse worldwide, but only about 20% of cases are known. The remaining 80% are unknown, but the general opinion is that they are likely caused by external factors. Diseases caused by external factors have too many causes to explain them with a single cause. Therefore, it is accepted that complex external factors are the main cause of breast cancer. Diseases caused by external factors cannot be predicted and prevented in advance, and treatment can only be done after diagnosis, which means that it takes a considerable amount of time in terms of cost, duration, and recovery. In the case of breast cancer, it is difficult to completely cure once cancer cells invade surrounding tissues or begin to metastasize to lymph nodes, so early detection is more important than other cancers, and research on this is actively underway (Republic of Korea Patent No. 10-1966493).

Breast cancer patients may experience bone loss during treatment and have a high risk of cancer cells metastasizing to bone. Between 65% and 75% of patients with metastatic breast cancer experience bone metastases.

Currently, bone metastases are diagnosed using X-rays, computed tomography, and magnetic resonance imaging. Biomarkers are being used clinically to diagnose and predict the prognosis of cancer patients in various types of cancer, but biomarkers related to bone metastases of breast cancer have not yet been used clinically.

Bone metastases are often diagnosed with a rapidly worsening prognosis regardless of the type of cancer, as the malignant tumor destroys bone tissue. The biggest reason for this is that the current diagnosis of bone metastases using imaging tests is very late. In fact, in breast cancer, which is prone to bone metastases, hormone suppression therapy is usually performed for 5 years after the initial treatment type, and “bone scans” are repeated at 6-12 month intervals for prevention and early diagnosis. However, even in cases where bone scans are diagnosed without symptoms such as pain, fractures, or nerve compression, bone destruction is often already advanced, and the treatment response is not good. In general, bone metastases progress through the dormant stage, microscopic bone metastases stage, and clinical bone metastases stage.

In the dormant stage, cancer cells exist only in bone cells, and there is no interaction between cancer cells and bone cells. However, in the microscopic bone metastasis stage, cancer cells interact with osteoclasts, and cell proliferation occurs. In the clinical bone metastasis stage, bone tissue is destroyed by cancer cells and osteoclasts, and symptoms such as pain and fractures are found. Conventionally, diagnosis of bone metastasis of cancer using imaging tests was possible only in this clinical bone metastasis stage, and in this case, effective treatment was difficult because the tumor had already progressed, and there was a limitation that diagnosis was possible after morphological changes in bone tissue occurred. Accordingly, there is an increasing need for a diagnostic composition, kit, or information providing method for diagnosis that can effectively treat through early diagnosis before bone tissue is destroyed in the microscopic bone metastasis stage.

Meanwhile, breast cancer cells with heterogeneity have different subtypes according to the expression patterns of various biomarkers, and are undergoing radiation therapy as well as drug therapy accordingly.

It is known that breast cancer subtypes according to the expression pattern of cells are divided according to the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). The type that has all three hormone receptors (ER+, PR+, HER2+) is called the luminal type, and drug treatment targeting the receptors is effectively being performed, but basal type breast cancer tissue that is negative for the three receptors (ER-, PR-, HER2-) is not only difficult to treat with drugs, but also has a high invasiveness and easy metastasis compared to the luminal type, showing a poor prognosis. Therefore, a new target that can treat metastatic breast cancer according to the above breast cancer cell expression pattern is needed.

Accordingly, the inventors of the present invention discovered that TOR1B is a biomarker for assessing bone metastasis in breast cancer patients, and in particular, confirmed that TOR1B can diagnose bone metastasis in ER+/PR+ breast cancer patients who are prone to bone metastasis.

Korean Patent No. 1966493 (2019.04.01)

The purpose of the present invention is to provide a diagnostic composition and kit capable of predicting or diagnosing bone metastasis in breast cancer patients.

In addition, the present invention aims to provide a method for providing information for predicting or diagnosing bone metastasis in breast cancer patients.

In order to solve the above problem, according to one aspect of the present invention, a composition for predicting or diagnosing bone metastasis of breast cancer is provided, which comprises a substance that specifically binds to Torsin Family 1 Member B (TOR1B) mRNA or its protein.

According to another aspect of the present invention, a kit for predicting or diagnosing bone metastasis of breast cancer comprising the composition is provided.

According to another aspect of the present invention, a method for providing information for predicting or diagnosing bone metastasis of breast cancer is provided, comprising the step of measuring the expression level of TOR1B mRNA or its protein in a sample isolated from a diagnostic subject.

The present invention relates to a composition and kit for predicting or diagnosing bone metastasis in breast cancer patients. The composition, kit, and method of the present invention can effectively predict and diagnose the bone metastasis status of breast cancer patients, and can significantly advance the timing of diagnosis of bone metastasis in breast cancer patients, thereby greatly improving the survival rate of patients.

Figure 1 shows the results confirming the localization of TOR1B at the cellular level ((A) COMPARTMENTS (B) Protein Atlas).
Figure 2 shows the results confirming that the mRNA expression of TOR1B was upregulated in bone metastasis patients in (A) E-MTAB-356 and (B) GSE2034 datasets.
Figure 3 is a diagram showing the ROC curve analysis to test the validity of TOR1B gene expression in determining bone metastasis and non-bone metastasis status of breast cancer patients through ROC curve analysis to predict bone metastasis in the datasets of (A) E-MTAB-356 and (B) GSE2034.
Figure 4 is a graph showing the Kaplan-Meier plot of TOR1B expression for bone metastasis based on the expression of TOR1B in breast cancer patients from (A) E-MTAB-356 and (B) GSE2034 datasets. (The p value was calculated by the log-rank test.)
Figure 5 is a graph confirming the significant association between TOR1B expression molecular markers and clinical information. The Kaplan-Meier curves are based on the expression of TOR1B in (A) ER-positive breast cancer patients from the E-MTAB-356 and (B) GSE2034 data sets, (C) PR-positive breast cancer patients, and (D) patients aged 55 years or older from the E-MTAB-356 data set. (The p value was calculated by the log-rank test.)
Figure 6 shows the multivariate Cox proportional hazards regression analysis of TOR1B and other clinical information.
Figure 7 is a diagram showing the interaction network of TOR1B with other genes and proteins. (A) The interaction network of TOR1B with other genes was generated using GeneMANIA, and (B) the interaction network of TOR1B with other genes was generated using STRING.

The present invention is described in detail below.

As used herein, the term "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of this application, diagnosis may mean confirming the presence or absence of bone metastases of cancer, or further confirming whether bone metastases of cancer have progressed or worsened.

The term "diagnosis" herein may include determining a subject's susceptibility to a particular disease or condition, determining whether a subject currently has a particular disease or condition, determining a prognosis for a subject having a particular disease or condition, or therametrics (e.g., monitoring a subject's condition to provide information about the efficacy of a treatment).

The present invention relates to a composition for predicting or diagnosing bone metastasis of breast cancer, comprising a substance that specifically binds to Torsin Family 1 Member B (TOR1B) mRNA or its protein.

TOR1B is an ATPase found primarily in the ER and nuclear envelope, and the protein product of the gene can act as a molecular chaperone that helps maintain proper folding of secretory or membrane proteins and membrane integrity (https://www.proteinatlas.org/ENSG00000136816-TOR1B).

TOR1B exists in the sample derived from the diagnostic subject, and its mRNA or protein sequence can utilize a sequence known in NCBI genbank, etc. For example, the sequence of the individual species being diagnosed can be used.

The above diagnostic subjects include animals that currently have breast cancer, animals that have had breast cancer, animals that wish to receive information for predicting or diagnosing bone metastasis of breast cancer, etc., and the animals may be mammals including humans.

The sample is separated from the diagnostic subject, and examples thereof include, but are not limited to, tissue, cells, blood, serum, plasma, saliva, or urine, and specifically, cancer tissue separated from the diagnostic subject.

The above material is not limited as long as it can detect TOR1B mRNA or its protein, but may be, for example, one from the group consisting of antibodies, aptamers, DNA, proteins, and polypeptides.

As used herein, "antibody" means a protein molecule specific for an antigenic site. For the purposes of this application, antibody means an antibody that specifically binds to the marker protein TOR1B, and may include monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.

The above monoclonal antibody may be produced using a hybridoma method or phage antibody library technology widely known in the art, but may not be limited thereto.

The above polyclonal antibodies can be produced by methods well known in the art, including injecting the above protein antigen into an animal and collecting blood from the animal to obtain serum containing antibodies. Such polyclonal antibodies can be produced from any animal species host, such as, but not limited to, goats, rabbits, sheep, monkeys, horses, pigs, cows, and dogs.

In addition, the antibodies of the present invention may also include special antibodies such as chimeric antibodies and humanized antibodies.

The above "peptide" has the advantage of high binding affinity to the target substance and does not undergo denaturation even when subjected to heat/chemical treatment. In addition, because it has a small molecular size, it can be used as a fusion protein by attaching to other proteins. Specifically, it can be used by attaching to a high molecular weight protein chain, so it can be used as a diagnostic kit and drug delivery material.

The above "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, an aptamer is composed of a polynucleotide that can specifically bind to an antigenic substance in the same way as an antibody, but is more stable than a protein, has a simple structure, and is easy to synthesize, and therefore can be used as a substitute for an antibody.

The substance that specifically binds to the above mRNA may be, but is not limited to, sense and antisense primers, or probes.

In the present invention, the term "primer" refers to a short genetic sequence that serves as the starting point of DNA synthesis, and an oligonucleotide synthesized for the purpose of diagnosis, DNA sequencing, etc. The primers are usually synthesized and used with a length of 15 to 30 base pairs, but this may vary depending on the intended use, and may be modified by methylation, capping, etc. using a known method.

In the present invention, the term "probe" refers to a nucleic acid capable of specifically binding to mRNA of several bases to several hundred bases in length, produced through an enzymatic chemical separation and purification process or a synthetic process. The presence or absence of mRNA can be confirmed by labeling with a radioactive isotope or enzyme, and can be designed and modified by a known method and used.

Since the nucleotide sequence of the gene encoding TOR1B is known, a probe or primer that specifically binds to the nucleotide sequence of the gene encoding TOR1B, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide can be designed by a person skilled in the art based on the sequence using a conventional method in the art.

The composition of the present invention is a material for use in predicting or diagnosing bone metastasis in breast cancer patients, and can be used to predict or diagnose bone metastasis in breast cancer patients by treating a sample isolated from a diagnosis subject and measuring the expression level of TOR1B mRNA or protein in the sample.

For example, if the expression level of TOR1B mRNA or its single protein in a sample of a patient with breast cancer bone metastasis is compared with the expression level of a normal control patient, and the expression level of the patient with breast cancer bone metastasis is higher than that of the normal control patient, it can be determined that the patient has a higher possibility of breast cancer bone metastasis than the normal control patient.

The present invention provides a kit for predicting or diagnosing breast cancer bone metastasis comprising the composition.

The kit may include not only a substance that specifically binds to TOR1B mRNA or its protein, but also one or more other component compositions, solutions or devices suitable for an analytical method for measuring the level of TOR1B mRNA or its protein expression utilized by the kit.

The above kit may be a kit including essential elements required for performing RT-PCR when it is a kit for measuring the expression level of TOR1B mRNA or protein. In addition to each primer pair specific for mRNA of a marker gene, the RT-PCR kit may include a test tube or other appropriate container, reaction buffer, deoxyribonucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC water, sterile water, etc. In addition, it may include a primer pair specific for a gene used as a quantitative control.

The above kit may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate for immunological detection of a nucleotide sequence of a gene encoding TOR1B, a sequence complementary to the above nucleotide sequence, a fragment of the above nucleotide, or a substance that specifically binds to a protein encoded by the above nucleotide sequence. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, a glass slide glass, etc., and the chromogenic enzyme may be peroxidase or alkaline phosphatase, the fluorescent substance may be FITC, RITC, etc., and the chromogenic substrate may be 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o-phenylenediamine (OPD), tetramethyl benzidine (TMB), etc.

The above kit may be a microarray for predicting or diagnosing breast cancer bone metastasis capable of measuring the expression level of TOR1B mRNA. The microarray may be easily manufactured by a person skilled in the art according to a method known in the art using the above indicator factor, and according to one specific example, it may be a microarray in which a cDNA of a sequence corresponding to an mRNA of a gene encoding the TOR1B protein or a fragment thereof is attached to a substrate as a probe.

In addition, the kit of the present invention may include an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a chromogenic substrate solution that reacts with the label to develop color, a washing solution, an enzyme reaction stop solution, etc., and may be manufactured with a plurality of separate packagings or compartments containing the reagent components used, but may not be limited thereto.

The kit of the present invention may include not only a preparation capable of measuring the expression level of TOR1B mRNA or its protein in a patient sample, but also one or more types of compositions, solutions or devices suitable for analyzing the expression level. For example, the kit may include, but is not limited to, a substrate, a suitable buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of antibodies.

As a specific example, the kit may be a kit characterized by including essential elements required to perform ELISA in order to implement various ELISA methods such as ELISA kits, sandwich ELISA, etc. The ELISA kit includes antibodies specific for the proteins. The antibodies have high specificity and affinity for TOR1B and little cross-reactivity to other proteins, and may be monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the ELISA kit may include antibodies specific for a control protein. In addition, the ELISA kit may include, but is not limited to, reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes, and substrates thereof, or other substances capable of binding to antibodies.

In addition, the kit may be a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each analysis method. Through these analysis methods, anticancer drug resistance can be diagnosed by comparing the amount of antigen-antibody complex formation.

The present invention provides a method for providing information for predicting or diagnosing breast cancer bone metastasis, comprising a step of measuring the expression level of TOR1B mRNA or its protein in a sample separated from a diagnostic subject.

The above measurement may be performed by treating the sample with a substance that detects TOR1B mRNA or its protein.

The substance detecting the above TOR1B mRNA or its protein may be within the range exemplified above.

The above samples and diagnostic subjects are as described above.

As a method for measuring the expression level of the above TOR1B mRNA or its protein, a method of measuring the concentration of mRNA, which is a transcript of a gene encoding TOR1B, in a sample or the concentration of the above TOR1B protein in a sample may be selected, but is not limited thereto, and a method commonly used in the technical field of the present invention may be selected and performed.

Methods for measuring the concentration of the mRNA in a sample include, but are not limited to, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chips.

As a method for measuring the concentration of the above protein in a sample, the amount of protein can be confirmed using an antibody that specifically binds to the protein. Analysis methods for this include, but are not limited to, an immunoblot test, a sandwich assay, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), western blotting, flow cytometry, enzyme-substrate colorimetry, antigen-antibody agglutination, and protein chips.

The information providing method of the present invention may further include a step of comparing the expression level with the expression level of a control group.

When the expression level of TOR1B mRNA or its protein in a sample of a breast cancer patient is compared with the expression level of a non-bone metastasis patient, if the expression level of TOR1B in the breast cancer patient is higher than that in the non-bone metastasis patient, it can be determined that the breast cancer patient has a high possibility of bone metastasis. In addition, if the expression level of the TOR1B mRNA or its protein is measured to be low or not statistically significantly different from that in the non-bone metastasis patient (ex. p<0.05), it can provide information that can be determined that the possibility of bone metastasis in the breast cancer patient is low.

Additionally, by comparing the expression levels of TOR1B mRNA or its protein in samples from two breast cancer patients, it can be determined that a patient with a higher expression level has a higher possibility of bone metastasis than a patient with a lower expression level.

In addition, when the expression level of TOR1B mRNA or its protein in the samples of breast cancer patients is compared with the expression level of patients with breast cancer bone metastasis, if there is no statistically significant difference (P<0.05) between the expression level of breast cancer patients and the expression level of patients with breast cancer bone metastasis, it can be determined that the possibility of breast cancer bone metastasis is high.

Hereinafter, the present invention will be described in detail by way of examples in order to specifically explain the present invention.

Example

1. Subcellular localization of TOR1B

(1) Experimental preparation

The subcellular localization of TOR1B was predicted and visualized using the COMPARTMENTS database (https://compartments.jensenlab.org). All data were retrieved and filtered by selecting gene types in Homo sapiens, and genes were additionally collected from other databases using their unique gene symbols.

(2) Experimental method

The protein expression level of TORB1 was determined using the subcellular atlas of the Human Protein Atlas database (https://www.proteinatlas.org/). The gene symbol was used to retrieve information from the database, and immunohistochemical (IHC) staining of TOR1B in the endoplasmic reticulum and nucleus was evaluated in the subcellular atlas.

(3) Experimental results

The subcellular localization of TOR1B was predicted using the COMPARTMENTS database, taking into account multiple sources of information, namely automatic text mining of biomedical literature, database annotations, and sequence-based predictions for various cell types. The TOR1B protein product was identified with highest confidence (confidence level 5) in the endoplasmic reticulum and nucleus, and with intermediate confidence (confidence level 4) in the extracellular space (p-value).

The lowest confidence (confidence level 1) was found in the cytoplasm, cytoskeleton, mitochondria, peroxisomes, endosomes, lysosomes, and Golgi apparatus (Fig. 1A). TOR1B protein expression was projected using the subcellular atlas from the Human Protein Atlas database, and was highly expressed in the endoplasmic reticulum and nucleus (Fig. 1B).

2. Gene expression profiles, clinical data and analysis

(1) Experimental method

Gene expression profiles and clinical information of breast cancer (BC) patients were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (www.ncbi.nlm. nih.gov/geo/) and ArrayExpress (https://www.ebi.ac.uk/arrayexpress/).

The robust multiarray average algorithm (RMA) was used to normalize the raw data to the median via Cluster 3.0. The GSE2034 and EMTAB-365 datasets, consisting of 286 and 425 patients, respectively, were used as training and validation cohorts. The probe set identifiers were converted to gene symbols. The gene expression levels were normalized to the median and classified into two expression groups. Patients with TOR1B expression levels higher than the median were considered as the high expression group, and patients with TOR1B expression levels below the median were considered as the low expression group. These groups were used for further analysis.

(2) Experimental results

The mRNA expression of TOR1B was evaluated between metastatic and non-metastatic breast cancer (BC) patients in the E-MTAB-365 and GSE2034 datasets. The upregulation of TOR1B was highly significant in bone metastasis (BM) patients ( p = 8e-04 and p = 4.5e-05, metastatic vs. non-metastatic breast cancer (BC), E-MTAB-365 and GSE2034 datasets, respectively) (Fig. 2). These results indicated that higher TOR1B expression at the transcript level may be associated with bone metastasis (BM) in breast cancer (BC) patients.

3. Correlation with TOR1B expression and clinical information

(1) Experimental method

To observe the mRNA expression level of TOR1B among patients with bone metastasis of breast cancer, a total of 711 patients were selected from the GSE2034 and E-MTAB-365 datasets. Based on the mRNA expression level, the Kaplan-Meier method was used to compare the survival between patients with bone metastasis (BM) and those without bone metastasis (BM), and patients with ER and PR positive BC. The Chi-square and log-rank tests were used to evaluate patient survival and metastasis.

(2) Experimental results

This study included 425 breast cancer (BC) patients from the E-MTAB-365 dataset and 286 breast cancer (BC) patients from the GSE2034 dataset. The associations between TOR1B expression and clinical features are shown in Table 1 . Based on the median expression of TOR1B, patients in the E-MTAB-365 dataset were classified into high expression (n=212) and low expression (n=213) groups.

To study the association between clinicopathologic features of breast cancer (BC) patients and TOR1B expression (high and low), a chi-square test was performed. The results showed that age and tumor grade of breast cancer (BC) patients were not significantly correlated with TOR1B expression (p>0.05). With regard to hormone receptor status, the presence or absence of HER2 was significantly associated with TOR1B expression ( p = 0.008). In addition, TP53 mutation was highly correlated with TOR1B upregulation (p=0.007), and CIT classification, TNM stage, and bone metastasis (BM) of breast cancer (BC) patients were significantly associated with TOR1B expression ( p = 5.00E-04, p = 0.023, and p = 0.001, respectively).

Table 1 below describes the clinicopathologic characteristics of the two groups of breast cancer patients based on the median expression of TOR1B.

Variable Total Low expression High expression p (χ2- test) Number of patients (%) 425 213 (50.1) 212 (49.9) Age ≤55 216 (51.3) 109 (25.9) 107 (25.4) 0.788 >55 205 (48.7) 100 (23.8) 105 (24.9) ER No 103 (24.8) 50 (12) 53 (12.8) 0.74 0.74 Yes 312 (75.2) 158 (38.1) 154 (37.1) PR No 167 (40.3) 87 (21) 80 (19.3) 0.538 Yes 248 (59.8) 121 (29.2) 127 (30.6) HER2 No 250 (85) 113 (38.4) 137 (46.6) 0.008 Yes 44 (15) 30 (10.2) 14 (4.8) Grade I 34 (8.2) 12 (2.9) 22 (5.3) 0.209 II 215 (51.5) 111 (26.6) 104 (24.9) III 168 (39.6) 86 (20.6) 82 (19) TP53 status Mutation 76 (50) 43 (28.3) 33 (21.7) 0.007 Wild type 76 (50) 60 (39.5) 16 (10.5) CIT basL 47 (11) 12 (2.8) 35 (8.2) 5.00E-04 lumA 81 (19) 49 (11.5) 32 (7.5) lumB 86 (20.2) 62 (14.6) 24 (5.6) lumC 62 (14.6) 34 (8) 28 (6.6) mApo 36 (8.5) 22 (5.2) 14 (3.3) normL 113 (26.6) 34 (8) 79 (18.6) TNM stage N0 134 (32.1) 79 (18.9) 55 (13.2) 0.023 N1 284 (67.9) 133 (31.8) 151 (36.1) Bone metastasis No 355 (83.5) 165 (38.8) 190 (44.7) 0.001 Yes 70 (16.5) 48 (11.3) 22 (5.2)

The sensitivity and specificity of TOR1B in predicting bone metastasis (BM) were evaluated from the area under the curve (AUC) of receiver operating characteristic (ROC) curve analysis. The AUC of TOR1B showed significant predictive ability for bone metastasis (BM) of breast cancer (BC) patients in both E-MTAB-365 and GSE2034 datasets (AUC=0.627, p<0.001 and AUC=0.663, p<0.001, respectively) (Fig. 3).

To study the prognosis of breast cancer (BC) patients with respect to TOR1B upregulation, we generated a Kaplan-Meier plot, and the results showed that patients with upregulated TOR1B expression developed bone metastasis (BM) earlier than those with low TOR1B expression (Fig. 4).

In the present invention, we further studied TOR1B expression in patients with bone metastasis (BM) using various clinicopathologic features, i.e., age, presence or absence of hormone receptors, etc., to identify patients most prone to develop early metastasis. The results demonstrated that TOR1B upregulation was significantly associated with early bone metastasis in ER+ (E-MTAB-365, p =0.0159 and GSE2034, p =0.00182) and PR+ (E-MTAB-365, p=0.00742) breast cancer (BC), and patients younger than 55 years old were more susceptible to early metastasis (E-MTAB-365, p =0.0182). In addition, we performed multivariate Cox proportional hazards regression analysis of TOR1B together with other clinical information to identify predictive factors in breast cancer (BC) patients. Results revealed that TOR1B was an independent predictor of bone metastasis (BM) in breast cancer (BC) patients ( p =0.024) (Figs. 5 and 6).

4. TOR1B gene-gene, protein-protein interaction network analysis

(1) Experimental method

The interactions between TOR1B gene and other genes were analyzed using the GeneMANIA database (https://genemania.org/). The gene interaction network was constructed based on the functional associations of genes present in the dataset, including protein and genetic interactions, signaling pathways, co-expression, co-localization, and protein domain similarity. The analysis parameters were set according to the default indices. Protein interaction (PPI) network analysis was performed using the Protein Interaction Gene/Protein Search (STRING) database (https://www.stringdb.org/).

The PPI network of TOR1B was investigated in the Homo sapiens database of STRING. The TOR1B protein network was established based on eight criteria: experimental evidence and curated databases, gene neighborhood, gene fusion, gene co-occurrence, text mining, co-expression, and protein homology.

(2) Experimental results

To study the interaction network of TOR1B with other proteins and genes, TOR1B was introduced into GeneMANIA and STRING databases and molecular network analysis was performed. TOR1B was closely associated with other proteins, such as NEDD8, TOR1A1P2, DYNLT1, TOR1A, TNFRSF25, PRPF4B, WDR11, STON2, TNFRSF10A, and COPS4 (Fig. 7).

In addition, among various Homo sapiens genes, TOR1B was confirmed to be related to TOR1A, TOR1A1P2, TOR1A1P1, TOR2A, TOR3A, TOR4A, BUB1, GIMAP2, GAPVD1, LIPC, GPR107, FBXO6, FUBP3, UBE201, LAGE3, EN2, FAM32A, SPTLO1, ERP44, and TOPORS genes (Fig. 3).

5. Statistical Analysis

Kaplan-Meier curves, chi-square tests, and log-rank tests were performed using the R programming language (www.r-project.org). The Wilcoxon signed-rank test was used to compare clusters between two groups. The predictive ability of the biomarkers was assessed by multivariate analysis. The sensitivity and specificity of the prognostic biomarkers were assessed by AUC, and a two-sided P value of less than 0.05 was considered statistically significant.

Claims (6)

A composition for predicting or diagnosing bone metastasis of breast cancer, comprising a substance that specifically binds to Torsin Family 1 Member B (TOR1B) mRNA or its protein.
A composition for predicting or diagnosing bone metastasis of breast cancer, wherein the material is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, and protein polypeptides according to claim 1.
A kit for predicting or diagnosing bone metastasis of breast cancer comprising the composition of claim 1 or 2.
A method for providing information for predicting or diagnosing bone metastasis of breast cancer, comprising a step of measuring the expression level of TOR1B mRNA or its protein in a sample isolated from a diagnostic subject.
A method for providing information for predicting or diagnosing bone metastasis of breast cancer, further comprising a step of determining that the diagnostic subject has a higher possibility of bone metastasis compared to the control group if the expression level is higher than the expression level of the control group in claim 4.
A method for providing information for predicting or diagnosing bone metastasis of breast cancer, wherein the sample according to claim 4 is selected from the group consisting of tissue, cell, blood, serum, plasma, saliva, and urine.