KR20180094497A - Method for predicting the response and prognosis to chemotherapy in a patient with triple-negative breast cancer using NR1D1 - Google Patents

Abstract

The present invention relates to a method for predicting response and prognosis to chemotherapy in a patient with triple-negative breast cancer using nuclear receptor subfamily 1, group D, member 1 (NR1D1), and as it was confirmed that chemotherapy prognosis is good in patients with high NR1D1 expression, in patients with the triple-negative breast cancer, the degree of expression of NR1D1 can be confirmed to predict the response and prognosis of chemotherapy. Therefore, the expression of NR1D1 in the patient with the triple-negative breast cancer is measured, and side effects of the chemotherapy can be reduced by controlling the dose of an anticancer drug depending on the degree of expression, and thus it is anticipated that it will be possible to develop a patient-oriented anticancer treatment strategy which has higher efficacy than existing anticancer therapy.

Description

[0001] The present invention relates to a method for predicting the response and prognosis of chemotherapy in a triple-negative breast cancer patient using NR1D1,

The present invention is an analysis of the correlation between NR1D1 expression level and overall survival rate after chemotherapy in breast cancer patients and measuring the expression level of NR1D1 (Nuclear receptor subfamily 1, group D, member 1) The present invention relates to a method of providing information for anticancer drug therapy response and prognosis prediction in triple-negative breast cancer patients.

Breast cancer is the most commonly diagnosed cancer in women and the fifth leading cause of cancer-related deaths worldwide. In general, the primary treatment for breast cancer patients is surgery, and subsequent adjuvant chemotherapy is treated to improve the patient survival rate by eradicating residual cancer cells. An anthracycline such as doxorubicin and epirubicin, an alkylating agent such as cyclophosphamide and taxanes such as paclitaxel and docetaxel include standard chemotherapy regimens for the treatment of breast cancer. Platinum-based drugs have also been considered as a component of combination therapy, particularly for the treatment of patients with triple-negative breast cancer (TNBC).

(ER), progesterone receptor (PR), and erb-b2 receptor tyrosine kinase 2 (ERBB2, also referred to as human epithelial growth factor receptor 2 (HER2)) are found in the breast cancer subtypes LUMINA A, LUMINAL B, ERBB2, And molecular biomarkers that distinguish triple-negative [Perou et al., 2000]. This triple negative breast cancer (TNBC) without ERα, PR, and ERBB2 expression is the most aggressive subtype with a histologic grade and poor clinical outcome [Reis-Filho and Pusztai, 2011]. Because of the lack of such traditional targets, there is no specific systemic treatment such as endocrine therapy and HER2 targeted therapy for TNBC. Currently, chemotherapy and radiation therapy are the basis of TNBC therapy, which is associated with serious side effects. Thus, identification of new targets will provide an advantage in the treatment of TNBC by minimizing side effects.

On the other hand, a method of providing information for determining the prognosis of breast cancer by predicting axillary lymph node metastasis of breast cancer has been disclosed (Korean Patent Laid-Open Publication No. 10-2015-0043898), but specifically, There has been no disclosure about therapeutic response and prognosis prediction.

On the other hand, despite the known efficacy of the components of the combination therapy described above, drug resistance frequently occurs in breast cancer patients, which is still a major obstacle to cancer treatment. Therefore, there is a need to develop new therapeutic strategies to reduce the resistivity and to improve the effectiveness of chemotherapy.

The present invention relates to a method for providing information for anticancer drug response and prognosis prediction in triple-negative breast cancer patients, comprising the step of measuring the expression level of NR1D1 (Nuclear receptor subfamily 1, group D, member 1) .

According to one aspect of the present invention there is provided a method of predicting the response and prognosis of chemotherapeutic treatment in a triple-negative breast cancer patient, comprising the step of measuring the expression level of NR1D1 (Nuclear receptor subfamily 1, group D, member 1) Provide information providing method.

According to another aspect of the present invention, there is provided a pharmaceutical composition for the triple-negative breast cancer patient, which comprises an agent capable of measuring the expression level of NR1D1 (Nuclear receptor subfamily 1, group D, member 1) Lt; / RTI >

According to another aspect of the present invention, there is provided a kit for predicting the response and prognosis of an anticancer drug in a triple-negative breast cancer patient, comprising the biomarker composition.

According to the present invention, the method of providing information for anticancer drug therapy response and prediction of prognosis of triple-negative breast cancer patients has good chemotherapy prognosis in patients with high NR1D1 expression, , The anticancer drug response and prognosis can be predicted by confirming the expression level of NR1D1.

Accordingly, the degree of expression of NR1D1 in triple-negative breast cancer patients can be measured, and the dose of the anticancer drug can be controlled according to the degree of expression, thereby reducing the side effects of the chemotherapy, It is expected that a treatment strategy will be established.

Figure 1 shows the results of experiments showing that high expression of NR1D1 correlates with improved clinical outcome in patients with breast cancer who received chemotherapy.
Figure 2 shows a representative case of immunohistological staining of NR1D1 in a breast cancer tissue microarray showing the immune response of a representative tumor sample. (The NR1D1-Low and NR1D1-High groups are defined in the method. Scale bar), 100 [mu] m).
FIG. 3 shows the significance of NR1D1 expression in triple-negative breast cancer patients treated with chemotherapy. The results of the Kaplan-Meier test for OS and DFS according to NR1D1 expression in triple-negative breast cancer patients treated with chemotherapy Respectively.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Nuclear receptor subfamily 1, group D, member 1 (NR1D1), also called REV-ERBa, is a transcription factor that directly binds to ligand regulatory nuclear receptors and specific DNA response elements and inhibits target gene transcription. NR1D1 regulates a variety of biological processes such as biological clocking, cell differentiation, metabolism, immune response and behavior. Recent studies have shown that NR1D1 is significantly associated with breast cancer. The NR1D1 gene is located at the ERBB2 amplification product site on chromosome 17q11-12 and has been proposed as part of the ERBB2 signal associated with poor clinical outcome in breast cancer. On the other hand, the pharmacological activation of NR1D1 reduced proliferation in breast cancer cells regardless of the molecular subtype of breast cancer. We recently examined the novel function of NR1D1 in the DNA damage repair pathway, which could be a potential therapeutic target for TNBC. In breast cancer cells, NR1D1 is recruited to DNA damage sites and interacts with poly (ADP-ribose) polymerase 1 (PARP1), thereby inhibiting the mobilization of DNA damage response complexes containing BRCA1. In addition, NR1D1 expression levels were positively correlated with clinical outcomes in breast cancer patients receiving chemotherapy. Although NR1D1 can function as a regulator of DNA repair and prognostic factors in TNBC, the potential as a therapeutic target for breast cancer has not been evaluated in clinical outcomes. Therefore, we retrospectively examined NR1D1 expression in breast cancer patients and assessed the potential prognostic value of NR1D1 expression. Analysis of four open patient datasets showed that NR1D1 expression was positively correlated with clinical outcome in breast cancer patients receiving chemotherapy. Our results indicate that NR1D1 and its ligands provide treatment options to improve the outcome of chemotherapy in breast cancer patients.

We suggest that NR1D1 can be used as a new predictive biomarker for breast cancer to facilitate a more personalized therapeutic approach.

A more detailed analysis of NR1D1 gene expression levels and changes in genes associated with DDR response can improve outcome prediction with DNA damage-inducing chemotherapy. In particular, patients with TNBC frequently have germline BRCA1 mutations and a variety of phenotypic traits in BRCA1-associated breast cancer, including mutations in BRCA1 silencing or other DNA repair-related genes such as p53. Thus, TNBC may be a major target for testing the hypothetical use of NR1D1 as a predictive biomarker for breast cancer chemotherapy to facilitate a more personalized therapeutic approach.

High NR1D1  Expression is associated with better clinical outcomes in breast cancer patients.

We analyzed gene expression profiles obtained from public data sets including clinical outcome information such as pathologic complete response (pCR), disease free survival (DFS), and recurrence free survival (RFS). pCR is an important indicator of improved chemosensitivity in solid tumors and is associated with favorable DFS and overall survival. Two independent data sets, GSE4056 and GSE34138, showed that the subgroup of patients with higher NR1D1 expression levels had a higher pCR ratio. The odds ratios indicating the effect of predictive variables were 3.75 for GSE4056 and 2.11 for GSE34138, indicating a positive correlation between NR1D1 expression levels and better clinical outcomes in breast cancer patients (Table 1). Consistent with these results, NR1D1 expression levels were higher in patients of the achieved pCR group than in patients of non-attained pCR group with statistical or marginal significance in these data sets. The Kaplan-Meier method was also used to analyze the survival rate by log rank testing on two independent data sets, GSE 1456 and NKI. RFS or DFS is enhanced in the high NR1D1 expression group to demonstrate the beneficial effect of chemotherapy on recurrence in patients with high NR1D1 expression levels (Fig. 1). Figure 1 shows that GSE 1456 and the NKI data set are located at the NCBI GEO site and Dr. Obtained from websites provided by Bernards and his colleagues, respectively. Patients were classified as low NR1D1 expression (low quartile) and high NR1D1 expression (high quartile) groups. The DFS or RFS ratio (%) is shown for each group. Log-rank (Mantel-Cox) tests were performed to analyze the statistical differences.

Many recent studies aimed to identify useful markers to reveal the outcome of chemotherapy. In this study, we found that breast cancer patients with high NR1D1 expression levels obtained better clinical outcomes after chemotherapy in the clinic (Table 1 and Figure 1). This observation can show that NR1D1 allows successful achievement of pCR in breast cancer patients. Achieving pCR with primary chemotherapy is very important for successful treatment of breast cancer patients, so careful consideration is needed to identify the best treatment options. We suggest that NR1D1 can be used as a new predictive biomarker for breast cancer to enable a more personalized therapeutic approach.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  One

Analysis of breast cancer patient populations based on public data sets (high NR1D1  Expression is associated with better clinical outcomes in breast cancer patients In related )

Public data sets GSE4056, GSE34138, and GSE1456 were downloaded from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/), and the NKI data set was generated by Dr. Bernards and his colleagues Obtained from the provided website (http://ccb.nki.nl/data/). The GSE4056 data set included gene expression profiles in a population of 100 patients, of which 44 in the validation set received gemcitabine, epirubicin, and docetaxel as basic standard chemotherapy. The GSE34138 data set included the gene expression profiles of 178 patients treated with single dose-concentration doxorubicin and cyclophosphamide chemotherapy. GSE1456 included the gene expression profiles of 159 patient groups who received intravenous cyclophosphamide, methotrexate, and 5-fluorouracil mainly as adjuvant chemotherapy. The NKI data set (Agilent chip with NKI annotated probe) included a gene expression profile of 295 patient populations, of which 110 patients who received chemotherapy for analysis were selected. Data lacking expression signals in the microarray or no clinical information records were excluded from all analyzes. The processed data including the normalization procedure was obtained from the corresponding website and no further transformation was performed.

We analyzed gene expression profiles from published data sets that included clinical outcome information such as pathologic complete response (pCR), disease free survival (DFS), and recurrence free survival (RFS). pCR is an important indicator of improved chemosensitivity in solid tumors and is associated with favorable DFS and overall survival. Two independent data sets, GSE4056 and GSE34138, showed that the subgroup of patients with higher NR1D1 expression levels had a higher pCR ratio. The odds ratios indicating the effect of predictive variables were 3.75 for GSE4056 and 2.11 for GSE34138, indicating a positive correlation between NR1D1 expression levels and better clinical outcomes in breast cancer patients (Table 1). Consistent with these results, NR1D1 expression levels were higher in patients of the achieved pCR group than in patients of non-attained pCR group with statistical or marginal significance in these data sets. The Kaplan-Meier method was also used to analyze the survival rate by log rank testing on two independent data sets, GSE 1456 and NKI. RFS or DFS is enhanced in the high NR1D1 expression group to demonstrate the beneficial effect of chemotherapy on recurrence in patients with high NR1D1 expression levels (Fig. 1).

Example  2

group On a microarray  Analysis of breast cancer patient group based on

NR1D1  Characteristics of Breast Cancer Patients by Expression

Primary breast cancer samples were obtained from Samsung Medical Center in Seoul, Korea from 1995 to 2002. All breast cancer patients were retrospectively studied. This breast cancer group was named 'Samsung Medical Center Breast Cancer Biomarker Research (SMC-BCBS)'. Clinical pathologic data including age, tumor characteristics (tumor size, lymph node involvement, LN metastasis, AJCC stage, and pathology stage), adjuvant chemotherapy treatment, and survival data were obtained from medical records (Table 2). The AJCC stage, which describes the extent of disease progression in cancer patients, was defined according to the American Joint Committee on Cancer criteria. The cases were divided into breast cancer subtypes, luminal A, luminal B, HER2, and TNBC. This medical record was approved by the Ethics Committee of the Institutional Review Board of Samsung Medical Center.

TMA sections were incubated with primary antibodies against NR1D1 (H00009572-M02, Novus Biologicals LCC, CO, USA) diluted 1: 1000 at room temperature for 1 hour. The tissue sections were then washed and reacted with an anti-mouse secondary antibody conjugated with HRP labeled polymer (K4001, DAKO, Denmark). The sections were then visualized using a high sensitivity substrate-dye source system (K3468, DAKO, Denmark). Control staining was performed with Meyer's hyalinoxacillin. Immunohistochemical staining was scored by a pathologist based on the intensity of the staining and the percentage of stained tumor cells. Immunohistochemical staining scores were as follows: score = 0, n = 33 (4.8%); Score = 1, n = 63 (9.1%); Score = 2, n = 112 (16.1%); Score = 3, n = 42 (6.1%); Score = 4, n = 123 (17.7%); Score = 6, n = 182 (26.2%); Score = 9, n = 139 (20.0%).

We have confirmed NR1D1 expression in primary invasive breast cancer tissue samples by immunohistochemistry. A total of 694 cases with beneficial immunohistochemistry results were included in this study. A representative tissue microarray stained for NR1D1 is shown in Fig. The signal of NR1D1 was found mainly in the nuclei of tumor cells, and substrate staining was not observed in almost all samples. Immunohistochemical staining scores were evaluated and classified into NR1D1-Low and NR1D1-High groups. Overall, a high NR1D1 expression group (score = 9, n = 139 (20.0%)), or a low NR1D1 expression group (score <9, n = 555 (80.0%)). Table 2 summarizes the clinicopathologic characteristics of patients in the study group. Most patients (87.5%) were treated with adjuvant chemotherapy. We estimated the correlation between NR1D1 expression and clinicopathologic factors. NR1D1 expression was significantly correlated with clinicopathologic characteristics for histologic grade (P = 0.011) and estrogen receptor (ER) status (P = 0.021). However, no differences were observed in the distribution of other parameters including adjuvant chemotherapy (Table 2).

Survival analysis

Correlation between NR1D1 expression and clinicopathologic characteristics was statistically analyzed using chi-square test. Overall survival (OS) was used to describe the time from the primary operation date to the date of death or the last follow-up date. Disease free survival (DFS) is defined as the period from primary surgery to local recurrence and distant metastasis, or recurrence, which means death from any cause.

The mean follow-up time for overall survival (OS) and disease free survival (DFS) was 10.3 years (range 0.1 to 19.5 years) and 9.4 years (range 0.1 to 15.5 years), respectively. During the follow-up period, 28.6% (198 of 692) of the patients recurred and / or metastasized, and 23.9% (165 of 691) of the patients died. To investigate the association of NR1D1 expression with clinical outcome, survival analysis was performed using the Kaplan-Meier method as a log-rank test. When subgroup analysis was performed on the molecular subtypes of breast cancer, high NR1D1 expression showed significantly improved OS (P = 0.008) and DFS (P = 0.023) in TNBC patients. In addition, a significant effect of NR1D1 expression on OS (P = 0.002) and DFS (P = 0.007) was significant in TNBC patients treated with chemotherapy (FIG. 3). These results demonstrate that high NR1D1 expression has a beneficial effect on OS and DFS in TNBC patients.

Prognostic factor analysis

The Kaplan-Meier method was used for survival analysis, and the log-rank test was used to estimate the association between the variables and survival. A univariate logistic regression model was used to estimate the association between clinicopathologic factors and NR1D1 expression. A multivariate Cox regression model was used to identify significant prognostic factors between clinicopathologic factors and NR1D1 expression. A P value of less than 0.05 was considered statistically significant. Statistical analysis was performed using software R for statistical calculations and graphics (http://r-project.org).

We assessed the association of NR1D1 expression with survival in TNBC patients receiving chemotherapy. Univariate analysis showed that patients with high NR1D1 expressing TNBC had a better OS and DFS than patients with lower NR1D1 expression after chemotherapy. The low OS and DFS predictors based on univariate analysis were higher tumor size, presence of lymph node involvement, presence of LN metastasis, and higher AJCC stage in patients with TNBC treated with chemotherapy (Table 3). To get deeper, multivariate analysis was performed using the Cox proportional hazards model for some of the factors that were found to be significant in the univariate analysis. Importantly, NR1D1 expression in TNBC patients receiving chemotherapy includes statistically significant improvements in OS and DFS in multivariate analysis. Our results show that high NR1D1 expression is associated with the benefits of chemotherapy and may be an independent prognostic factor in TNBC patients (Table 4).

*High NR1D1 Expression ^a of  With breast cancer patients Increased  Therapeutic response

^a GSE4056 and GSE34138 data sets with therapeutic response information were obtained from the NCBI GEO site.

^b patients were classified into the low (sub-averaged) NR1D1 expression group and the high (average) NR1D1 expression group.

^c Fisher's exact probability test was performed for significance verification by a web-based program (http://vassarstats.net/). CI, confidence interval.

Characteristic Total patient NR1D1 -Low NR1D1 -High
P- value ^a n = 694 n = 555 n = 139 No. (%) No. (%) No. (%) Age at diagnosis (years)
≤50
> 50
420
274
60.5
39.5
343
212
61.8
38.2
77
62
55.4
44.6 0.199 Tumor size
T1
T2
T3 or T4
269
378
47
38.8
54.5
6.8
221
292
42
39.8
56.2
7.6
48
86
5
34.5
61.9
3.6 0.077 Lymph node involvement
N0
N1
N2
N3
354
180
85
75
51.0
25.9
12.2
10.8
288
142
67
58
51.9
25.6
12.1
10.5
66
38
18
17
47.5
27.3
12.9
12.2 0.813 LN transition
voice
positivity
354
340
51.0
49.0
288
267
51.9
48.1
66
73
47.5
52.5 0.404 AJCC stage
Ⅰ
Ⅱ
Ⅲ
168
353
173
24.2
50.9
24.9
142
275
138
25.6
49.5
24.9
26
78
35
18.7
56.1
25.2 0.210 Histological grade
One
2
3
Unknown
76
246
323
49
11.0
35.4
46.5
7.1
69
201
246
39
12.4
36.2
44.3
7.0
7
45
77
10
5.0
32.4
55.4
0.0 0.011 Estrogen receptor
voice
positivity
Unknown
282
411
One
40.6
59.2
0.1
213
341
One
38.4
61.4
0.2
69
70
0
49.6
50.4
0.0 0.021 Progesterone receptor
voice
positivity
Unknown
379
314
One
54.6
45.2
0.1
298
256
One
53.7
46.1
0.2
81
58
0
58.3
41.7
0.0 0.393 HER2 amplification
voice
positivity
504
190
72.6
27.4
411
144
74.1
25.9
93
46
66.9
33.1 0.113 Breast cancer subtype
Luminale
Luminal B
HER2
TNBC
Undefined
334
97
92
170
One
48.1
14.0
13.3
24.5
0.1
281
74
69
130
One
50.6
13.3
12.4
23.4
0.2
53
23
23
40
0
38.1
16.5
16.5
28.8
0.0 0.067 Adjuvant chemotherapy
Not receiving
Reception
Unknown
85
607
2
12.2
87.5
0.3
70
483
2
12.6
87.0
0.4
15
124
0
10.8
89.2
0.0 0.649

* NR1D1  Characteristics of Breast Cancer Patients by Expression

^a Chi square test, P value less than 0.05 is considered significant and is shown in bold.

variable OS DFS No. HR 95% CI P -value HR 95% CI P -value NR1D1 expression
lowness
height
123
37
1.00
0.09

0.01-0.63

0.016
1.00
0.17

0.04-0.73

0.017 Age at diagnosis
≤50
> 50
114
50
1.00
1.25

0.62-2.55

0.535
1.00
0.80

0.37-1.71

0.564 Tumor size
T1
T2
T3 or T4
56
93
11
1.00
2.16
7.63

0.87-5.36
2.46-23.70

0.096
<0.001
1.00
2.09
4.58

0.89-4.86
1.34-15.68

0.089
0.015 Lymph node involvement
N0
N1
N2
N3
94
36
17
13
1.00
1.03
3.01
15.26

0.36-2.94
1.13-8.04
6.46-36.05

0.950
0.028
<0.001
1.00
0.77
2.18
7.99

0.28-2.10
0.85-5.57
3.23-19.79

0.607
0.105
<0.001 LN transition
voice
positivity
94
66
1.00
2.79

1.37-5.66

0.005
1.00
1.77

0.90-3.46

0.099 AJCC stage
Ⅰ
Ⅱ
Ⅲ
43
84
33
1.00
1.55
6.65

0.50-4.74
2.22-19.93

0.446
<0.001
1.00
1.64
4.49

0.60-4.48
1.60-12.63

0.333
0.004 Histological grade
One
2
3
Unknown
3
31
100
26
-
1.00
1.86
-


0.64-5.39
-


0.251
-
-
1.00
1.57
-


0.60-4.14
-


0.358
-

* Who received chemotherapy TNBC  Patient OS and DFS Univariate  analysis

HR, hazard ratio: CI, confidential interval. A P-value less than 0.05 is considered significant and is shown in bold.

variable OS DFS No. HR 95% CI P -value HR 95% CI P -value NR1D1 expression
lowness
height
123
37
1.00
0.09

0.01-0.65

0.017
1.00
0.16

0.04-0.68

0.013 Tumor size
T1
T2
T3 or T4
56
93
11
1.00
0.76
1.05

0.14-4.13
0.15-7.37

0.753
0.963
1.00
0.73
0.76

0.14-3.79
0.10-5.65

0.705
0.793 LN transition
voice
positivity
94
66
1.00
0.97

0.27-3.57

0.967
1.00
0.64

0.19-2.19

0.474 AJCC stage
Ⅰ
Ⅱ
Ⅲ
43
84
33
1.00
2.36
8.61

0.30-18.35
0.52-144.06

0.413
0.134
1.00
3.02
10.53

0.43-21.03
0.71-157.21

0.265
0.088

* Chemotherapy recipients TNBC  Multivariate analysis of OS and DFS in patients

HR, hazard ratio: CI, confidential interval. A P-value less than 0.05 is considered significant and is shown in bold.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (5)

And determining the level of expression of the NR1D1 (Nuclear receptor subfamily 1, group D, member 1) in triple-negative breast cancer patients.
The method according to claim 1, wherein, when the NR1D1 expression level is high, the anticancer drug reactivity is predicted to increase.
The method according to claim 1, wherein, when the expression level of NR1D1 is high, it is predicted that the prognosis of chemotherapy treatment is improved.
A biomarker composition for predicting the response and prognosis of an anticancer drug treatment in a triple-negative breast cancer patient, comprising an agent capable of measuring the expression level of NR1D1 (Nuclear receptor subfamily 1, group D, member 1).
A kit for predicting the response and prognosis of chemotherapeutic treatment in triple-negative breast cancer patients, comprising the composition of claim 4.

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