WO2019095541A1

WO2019095541A1 - Composition and method for diagnosing and predicting breast cancer bone metastases

Info

Publication number: WO2019095541A1
Application number: PCT/CN2018/072269
Authority: WO
Inventors: 李宜健; 薛守海
Original assignee: 李宜健; 薛守海
Priority date: 2017-11-17
Filing date: 2018-01-11
Publication date: 2019-05-23

Abstract

Provided are a composition and method for diagnosing and predicting breast cancer bone metastases. At present, radionuclide bone scans (ECT), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography-computed tomography (PET-CT), and osseous tissue biopsy are gold standards for the discovery and diagnosis of breast cancer bone metastases. However, these methods all have different deficiencies, such as high examination costs, an increase in burden of patents due to interventional diagnosis, and an increase in pressure on routine examination of bone metastases in breast cancer patients. By studying and comparing the differences in gene expression in serum of breast cancer patients with and without bone metastases, gene markers that can be used for diagnosing and predicting different molecular subtypes of breast cancer bone metastases have been discovered and verified, and therefore, a composition and method capable of quickly diagnosing and predicting different molecular subtypes of breast cancer bone metastases by means of blood can be provided.

Description

Composition for diagnosing bone metastasis of breast cancer and diagnostic prediction method

Technical field

The invention belongs to the field of biochemistry, relates to a disease diagnosis composition and a diagnosis method, and particularly relates to a composition for diagnosing bone metastasis of breast cancer and a diagnostic prediction method.

Background technique

Breast cancer is one of the malignant tumors that threaten women's life and health. About 400-45 million people die of breast cancer every year. (Reference: Clinical characteristics and prognosis of patients with different molecular subtypes of breast cancer with bone metastases, Journal of Xi'an Jiaotong University· Medical Edition, September 2017, Vol. 38, No. 5). Breast cancer is very prone to distant metastasis. Bone is the most common distant metastasis site for breast cancer. More than 50% of patients have a metastatic tissue at the first metastatic site (Reference: Genes associated with breast cancer metastatic to bone, J Clin Oncol, 2006). Implications of Bone-Only Metastases in Breast Cancer: Favorable Preference with Excellent Outcomes of Hormone Receptor Positive Breast Cancer, Cancer Res Treat, 2011). According to the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2), Breast cancer is divided into four subtypes: Luminal A, Luminal B, Her-2 overexpression and triple negative breast cancer (Reference: Gene expression patterns of breast carcinomas) tumor subclasses with clinical implications, PNAS, 2001). Studies have shown that the prognosis of breast cancer bone metastasis is closely related to its molecular typing, clinical stage, lymph node status, etc. (References and risk factors of bone metastasis and skeletal related events in patients with primary breast cancer in Japan, Int J Clin Onco , 2014). The specific molecular biology and clinicopathological features of different molecular subtypes of breast cancer determine the difference in treatment and prognosis. There are also differences in gene expression levels of bone metastases in different molecular subtypes of breast cancer.

Early diagnosis of breast cancer bone metastasis is the key to saving patients' lives. At present, radionuclide bone scan (ECT), computed tomography (CT), nuclear magnetic resonance (MRI), positron emission tomography (PET-CT) and bone biopsy are the gold for the discovery and diagnosis of breast cancer bone metastasis. standard. However, these methods have different deficiencies, such as high inspection costs, and interventional diagnosis increases the burden on patients. This increases the pressure for routine testing of bone metastases in breast cancer patients.

Applicants aimed to study the differences in gene expression in serum of breast cancer patients with and without bone metastases. The findings and validations can be used as a genetic marker for the diagnosis and prediction of bone metastases in different molecular subtypes of breast cancer to provide a Kits and methods for rapidly diagnosing and predicting bone metastasis of different molecular subtypes of breast cancer by blood.

Summary of the invention

The object of the present invention is to overcome the deficiencies of the prior art and provide a lncRNA diagnostic composition, which is prepared as a diagnostic kit for detecting low-cost, non-invasive, convenient and rapid diagnosis of bone metastasis of different molecular subtypes of breast cancer.

The above object of the present invention is achieved by the following technical scheme (part of the lncRNA composition):

First, Luminal type A breast cancer bone metastasis

A lncRNA diagnostic composition consisting of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1.

The above diagnostic composition is useful in the preparation of a diagnostic kit for predicting bone metastasis of Luminal A breast cancer.

A diagnostic kit for the diagnosis of bone metastases in Luminal type A breast cancer, including qPCR primers for lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1.

Preferably, the diagnostic kit further comprises a qPCR primer of the internal reference GAPDH.

An enzyme required for qRT-PCR is also included in the diagnostic kit of any of the above.

A method for diagnosing bone metastasis of Luminal A breast cancer, comprising the following steps:

Step S1, collecting fasting venous blood of Luminal type A breast cancer patient, and separating the serum by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X ₃ , X ₁ , X ₂ ;

Step S3, substituting X ₃ , X ₁ , and X ₂ values into a binary logistic regression equation Y=-2.577+2.045X ₁ +1.956X ₂ +1.676X _{3 to} obtain a Y value, and a Y value greater than 0.598 indicates that the breast cancer patient develops bone. Metastasis, less than 0.598, indicates that no bone metastasis has occurred.

Second, Luminal B type breast cancer bone metastasis

A lncRNA diagnostic composition consisting of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452.

The above diagnostic composition is useful in the preparation of a diagnostic kit for predicting bone metastasis of Luminal B breast cancer.

A diagnostic kit for the diagnosis of bone metastases predicting Luminal B breast cancer, including qPCR primers for lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452.

The diagnostic kit of any of the above includes the enzyme required for qRT-PCR.

A method for diagnosing bone metastasis of Luminal B breast cancer, comprising the following steps:

Step S1, collecting fasting venous blood of Luminal B type breast cancer patient, and separating the serum by centrifugation after natural coagulation;

Step S2, extracting total serum RNA, and determining the relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in total RNA relative to the internal reference GAPDH by qRT-PCR, represented by X ₃ , X ₁ , X ₂ ;

Step S3, substituting X ₃ , X ₁ , and X ₂ values into a binary logistic regression equation Y=Y=-2.241+1.883X ₁ +2.275X ₂ +1.975X _{3 to} obtain a Y value, and the Y value is greater than 0.607, indicating that the breast cancer patient Bone metastasis occurred, less than 0.607 indicates no bone metastasis.

Third, Her-2 overexpression breast cancer bone metastasis

A lncRNA diagnostic composition consisting of lncRNA AK043773 and lncRNA EXOC7.

The above diagnostic composition is useful in the preparation of a diagnostic kit for predicting bone metastasis of Her-2 overexpressing breast cancer.

A diagnostic kit for the diagnosis of bone metastases in Her-2 overexpressing breast cancer, including qPCR primers for lncRNA AK043773 and lncRNA EXOC7.

A method for diagnosing bone metastasis of Her-2 overexpressing breast cancer comprises the following steps:

In step S1, the fasting venous blood of the Her-2 overexpressing breast cancer patient is collected, and the serum is separated by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X ₁ and X ₂ ;

Step S3, substituting X ₁ and X ₂ values into a binary logistic regression equation Y=-2.918+2.618X ₁ +2.115X _{2 to} obtain a Y value, and a Y value greater than 0.495 indicates that the breast cancer patient has bone metastasis, and less than 0.495 indicates that no occurrence occurs. Bone metastasis.

Four or three negative breast cancer bone metastasis

A lncRNA diagnostic composition consisting of lncRNA Lnc01089 and lncRNA HOTAIR.

The above diagnostic composition is useful in the preparation of a diagnostic kit for predicting bone metastasis of triple negative breast cancer.

A diagnostic kit for diagnosing bone metastasis predicting triple-negative breast cancer, including qPCR primers for lncRNA Lnc01089 and lncRNA HOTAIR.

A method for diagnosing bone metastasis of a triple negative breast cancer comprising the following steps:

Step S1, collecting fasting venous blood of a triple-negative breast cancer patient, and separating the serum by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X ₁ and X ₂ ;

Step S3, substituting X ₁ and X ₂ values into a binary logistic regression equation Y=-2.537+2.793X ₁ +2.181X _{2 to} obtain a Y value, and a Y value greater than 0.633 indicates that the breast cancer patient has bone metastasis, and less than 0.633 indicates that no occurrence occurs. Bone metastasis.

The above object of the present invention is achieved by the following technical scheme (methylated gene composition part):

First, Luminal type A breast cancer bone metastasis

A methylation gene diagnostic composition consisting of methylated PITX1 and methylated AMOT.

A diagnostic kit for the diagnosis of bone metastases in Luminal A breast cancer, including PCR amplification primers for methylated PITX1 and methylated AMOT.

Preferably, the diagnostic kit further comprises a pyrosequencing primer for methylated PITX1 and methylated AMOT.

Preferably, the diagnostic kit further includes enzymes and reagents required for PCR amplification and pyrosequencing.

Step S2, extracting total serum DNA, and determining methylation index of methylated PITX1 and methylated AMOT in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X ₁ and X ₂ ;

Step S3, substituting the values of X ₁ and X ₂ into the binary logistic regression equation Y=1/[1+EXP(1.499X ₁ +2.302X ₂ -0.258)] to obtain a Y value, and the Y value is less than 0.238, indicating that the breast cancer patient occurs. Bone metastasis, greater than 0.238, indicates no bone metastasis.

Second, Luminal B type breast cancer bone metastasis

A methylation gene diagnostic composition consisting of methylated PTPN1 and methylated SLIT2.

A diagnostic kit for the diagnosis of bone metastases in Luminal B breast cancer, including PCR amplification primers for methylated PTPN1 and methylated SLIT2.

Preferably, the diagnostic kit further comprises a pyrosequencing primer for methylated PTPN1 and methylated SLIT2.

Step S2, extracting total serum DNA, and determining methylation index of methylated PTPN1 and methylated SLIT2 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X ₁ and X ₂ ;

Step S3, substituting the values of X ₁ and X ₂ into the binary logistic regression equation Y=1/[1+EXP(2.016X ₁ +1.898X ₂ -0.455)] to obtain a Y value, and the Y value is less than 0.310, indicating that the breast cancer patient occurs Bone metastasis, greater than 0.310, indicates no bone metastasis.

Third, Her-2 overexpression breast cancer bone metastasis

A methylation gene composition consisting of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1.

A diagnostic kit for the diagnosis of bone metastases in Her-2 overexpressing breast cancer, comprising PCR amplification primers and pyrosequencing primers for methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1.

Step S2, extracting total serum DNA, and determining methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, in turn X ₁ , X ₂ , X ₃ ;

Step S3, substituting X ₁ , X ₂ , and X ₃ into the equation Y=1/[1+EXP(1.342X ₁ +1.401X ₂ +1.345X ₃ -2.035)] to obtain a Y value, and the Y value is less than 0.308, indicating that the breast cancer Bone metastasis occurred in the patient, and greater than 0.308 predicted no bone metastasis.

Four or three negative breast cancer bone metastasis

A methylation gene diagnostic composition consisting of methylated MYLK3 and methylated SCARA5.

A diagnostic kit for diagnosing bone metastasis predicting triple-negative breast cancer, including PCR amplification primers for methylated MYLK3 and methylated SCARA5.

Preferably, the diagnostic kit further comprises a pyrosequencing primer for methylated MYLK3 and methylated SCARA5.

Step S2, extracting total serum DNA, and determining methylation index of methylated MYLK3 and methylated SCARA5 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X ₁ and X ₂ ;

Step S3, substituting the values of X ₁ and X ₂ into the binary logistic regression equation Y=1/[1+EXP(1.775X ₁ +1.236X ₂ -0.398)] to obtain a Y value, and the Y value is less than 0.366, indicating that the breast cancer patient occurs. Bone metastasis, greater than 0.366, indicates no bone metastasis.

DRAWINGS

Figure 1 shows the ROC curve of the combination of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 for the diagnosis of bone metastases in Luminal A breast cancer unmetastatic and Luminal A breast cancer;

Figure 2 shows the accuracy of the combination of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in the diagnosis of bone metastases in Luminal A breast cancer unmetastatic and Luminal A breast cancer;

Figure 3 is a ROC curve of the combination of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in the test set for the diagnosis of bone metastases in Luminal B breast cancer unmetastatic and Luminal B breast cancer;

Figure 4 shows the accuracy of the combination of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in the diagnosis of bone metastases in Luminal B breast cancer unmetastatic and Luminal B breast cancer;

Figure 5 is a ROC curve of the combination of lncRNA AK043773 and lncRNA EXOC7 in the test set for the diagnosis of bone metastases in Her-2 overexpressing breast cancer unmetastatic and Her-2 overexpressing breast cancer;

Figure 6 shows the accuracy of the combination of lncRNA AK043773 and lncRNA EXOC7 in the diagnosis of bone metastases in Her-2 overexpressing breast cancer unmetastatic and Her-2 overexpressing breast cancer;

Figure 7 is a ROC curve for the diagnosis of bone metastases in triple-negative breast cancer unmetastatic and triple-negative breast cancer using a combination of lncRNA Lnc01089 and lncRNA HOTAIR in the test set;

Figure 8 shows the accuracy of the combined diagnosis of lncRNA Lnc01089 and lncRNA HOTAIR in the diagnosis of triple-negative breast cancer unmetastatic and triple-negative breast cancer bone metastases;

Figure 9 is a ROC curve of the combination of methylated PITX1 and methylated AMOT in the test for the differential diagnosis of Luminal A breast cancer unmetastatic and Luminal A breast cancer bone metastases;

Figure 10 is a graph showing the accuracy of the combination of focused methylated PITX1 and methylated AMOT for the diagnosis of bone metastases in Luminal type A breast cancer without metastasis and Luminal type A breast cancer;

Figure 11 shows the ROC curve of the combination of methylated PTPN1 and methylated SLIT2 in the test for the differential diagnosis of Luminal B breast cancer unmetastatic and Luminal B breast cancer bone metastases;

Figure 12 is a graph showing the accuracy of the combination of methylated PTPN1 and methylated SLIT2 for the diagnosis of bone metastases in Luminal B breast cancer without metastasis and Luminal B breast cancer;

Figure 13 is a ROC curve of the combination of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in the test for differential diagnosis of bone metastases in Her-2 overexpressing breast cancer unmetastatic and Her-2 overexpressing breast cancer;

Figure 14 is a graph showing the accuracy of the combination of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in the diagnosis of bone metastases in Her-2 overexpressing breast cancer unmetastatic and Her-2 overexpressing breast cancer;

Figure 15 is a ROC curve of the combination of methylated MYLK3 and methylated SCARA5 in the test combination for the diagnosis of bone metastases in triple-negative breast cancer unmetastatic and triple-negative breast cancer;

Figure 16 is a graph showing the accuracy of the combination of methylated MYLK3 and methylated SCARA5 in the diagnosis of triple-negative breast cancer unmetastatic and triple-negative breast cancer.

Detailed ways

The substantial content of the present invention is specifically described below with reference to the accompanying drawings and embodiments, but does not limit the scope of the present invention.

Part I: lncRNA diagnostic composition and diagnostic method

All breast cancer samples of this project were taken from September 2014 to September 2017 to the Nantong University Affiliated Hospital or Nantong First People's Hospital or Nanjing Drum Tower Hospital for the diagnosis of breast cancer without other malignant tumors. All samples were divided into Luminal A type, Luminal B type, Her-2 over-expressed type and triple negative type according to immunohistochemical detection. Various molecular subtypes were classified into breast cancer non-metastasis group and breast cancer bone metastasis group according to whether or not metastasis was performed. And each case of breast cancer bone metastasis group belongs to the bone as the first distant metastasis site. The breast cancer non-metastasis group and the breast cancer bone metastasis group were subjected to radionuclide bone scan (ECT), computed tomography (CT), nuclear magnetic resonance (MRI), positron emission computed tomography (PET-CT) and/or tissue. A biopsy and other tests confirmed. There was no significant difference in age between breast cancer non-metastasis group and bone metastasis group in each molecular subtype, which was comparable. Finally, each group of samples is randomly divided into a test set and a verification set.

All sample grouping information and sample numbers are diagnosed by the above gold standard as shown in the following table:

Collection of serum samples: Collecting 5.0 mL of fasting venous blood of the patient, centrifugation (4000 r/min, 2860×g) after natural coagulation, and separating the serum for 7 min, and storing at -80 °C for detecting the relative expression of target lncRNA in serum. .

Example 1: Luminal type A breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

Test set and validation set of Luminal type A breast cancer non-metastasis group, Luminal type A breast cancer bone metastasis group.

2. RNA extraction and qRT-PCR

Total RNA was extracted from serum samples using Trizol reagent (Invitrogen, Shanghai, China). The concentration and purity of total RNA were measured at 260 nm using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific), and electrophoretic detection revealed that the purified RNA was of good quality and then subjected to subsequent operations. Total RNA was converted to cDNA using a reverse transcription kit (Takara, Dalian, China). Fluorescence quantitative PCR (Takara, Dalian, China) was performed using the SYBR Green method, and data collection was performed using an ABI Prism 7000 fluorescent quantitative PCR system (Agilent Technologies). The primer was synthesized by Beauchamps (Shanghai, China), lncRNA XLOC_004122 primer sequence: upstream 5'-CTGGCAGGAACACCGGGTACTT-3', downstream 5'-TGACTTTTACTTAGGAGCCACTTCTTG-3'; lncRNA SUMO1P3 primer sequence: upstream 5'-CTGGAACTGGGAATGGAGGAAGA-3', downstream 5 '-GATTGAGAAAGGATTGAGGGAAA-3'; lncRNANBAT-1 Primer sequence: upstream 5'-CTGGGAAAGCCTGTGCTCTTGGA-3', downstream 5'-GCTTCACAGTGCTGCTCAATCGT-3'; GAPDH primer sequence: upstream 5'-CGCTCTCTGCTCCTCCTGTTC-3', downstream 5'-ATCCGTTGACTCCGACCTTCAC- 3'. LncRNA SUMO1P3 NBAT-1 and the relative expression of lncRNA average of 3 measurements was calculated by method ² -ΔΔCt lncRNAXLOC_004122,.

3, statistical processing

The data was analyzed using SPSS 19.0. The measurement data were expressed as mean ± deviation, using t test, the count data was expressed as a percentage, using χ ² test, P<0.05 was considered statistically significant, and the ROC curve was established to calculate the area under the curve (area under the curve, AUC). ) and 95% confidence interval. Logistic regression was used to screen the variables, and regression equations were established to generate a new set of variables Y. The ROC curve analysis was performed on the new variables and each individual indicator.

Second, the experimental results

1. Relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in Luminal type A breast cancer without metastasis and bone metastasis

In the test set, the relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in each sample were determined. Compared with the Luminal type A breast cancer non-metastasis group, the relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 were significantly up-regulated in the Luminal A breast cancer bone metastasis group. The bone metastasis group lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT- 1 The relative expression levels were (1.9 ± 0.4) times, (2.3 ± 0.5) times, and (2.5 ± 0.4) times the relative expression levels of the untransferred group, respectively.

2, lncRNA XLOC_004122, lncRNA SUMO1P3 or lncRNA NBAT-1 relative expression level alone for diagnosis and differentiation of Luminal A breast cancer without metastasis and Luminal A breast cancer bone transfer ROC curve analysis

The principle of the ROC curve evaluation method:

The basic evaluation indicators of the diagnostic test are sensitivity and specificity, and the comprehensive evaluation indicators include Youden index, ROC, AUC, and the like. For the evaluation of diagnostic tests, it is first necessary to know the true group of samples to be tested by the gold standard. For the disease group (corresponding to the breast cancer bone metastasis group in the present invention) and the health group (corresponding to the breast cancer non-metastasis group in the present invention) determined by the gold standard, the results of the diagnostic test can be classified into the following cases:

Positive (True Positive, TP); diagnostic test is positive (consistent with the gold standard results);

Negative (True Negative, TN); diagnostic test is negative (consistent with the gold standard results);

False Positive (FP): The test is positive in the diagnostic test (inconsistent with the gold standard results);

False Negative (FN): Negative in the diagnostic test (inconsistent with the gold standard results).

Can be expressed in the following table:

Sensitivity of the diagnostic test = A / (A + C); specificity of the diagnostic test = D / (B + D). Sensitivity and specificity can be used to determine the diagnostic sensitivity and specificity of diagnostic tests relative to the gold standard. High sensitivity means that the number of cases diagnosed as negative is small, and the rate of missed diagnosis is low; the high specificity means that the number of healthy cases is less positive and the rate of misdiagnosis is low.

The ROC curve is a curve based on the above sensitivity and specificity. Using the possible diagnostic thresholds in the diagnostic test as the diagnostic point, the corresponding sensitivity and specificity are calculated according to the above table. Then, with the sensitivity as the ordinate and the 1-specificity as the abscissa, the sensitivity and specific points of each point in each diagnostic point are marked in the coordinate map, and the coordinate points are connected to obtain a smooth curve, which is the ROC curve. . The more dense the diagnostic points are set, the smoother the resulting ROC curve will be.

The ROC curve uses each test result as a possible diagnostic threshold. The size of the area under the curve AUC indicates the accuracy of the diagnostic test. The area under the ROC curve AUC has been widely recognized as an intrinsic accuracy index for the authenticity evaluation of diagnostic tests. When the AUC is 0.5, there is no diagnostic significance; when the AUC is 0.5-0.7, the diagnostic accuracy is low; the AUC is 0.7-0.9. Time indicates that the diagnostic accuracy is medium; when AUC>0.9, the diagnosis has higher accuracy.

The relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 or lncRNA NBAT-1 were mapped in SPSS 19.0 alone to diagnose the ROC curves of Luminal A breast cancer unmetastatic and Luminal A breast cancer bone metastases, AUC were 0.601, 0.697, 0.729, respectively. , with low or medium accuracy.

3, lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 combined expression model construction and ROC curve analysis for diagnosis of Luminal A breast cancer non-metastasis and Luminal A breast cancer bone metastasis

The relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in the test set samples were used as independent variables (X ₁ = lncRNA SUMO1P3 relative expression level, X ₂ = lncRNA NBAT-1 relative expression level, X ₃ = lncRNA XLOC_004122 relative Expression level), as a dependent variable by group (ie, whether the sample belongs to the bone metastasis group or the non-metastasis group according to the gold standard), for lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in Luminal type A breast cancer without metastasis and Luminal type A Binary logistic regression was performed on the relative expression levels in breast cancer bone metastasis samples to obtain a binary logistic regression equation: Y=-2.577+2.045X ₁ +1.956X ₂ +1.676X ₃ ; then lncRNA XLOC_004122, lncRNA SUMO1P3 in each sample Substituting the relative expression level of lncRNA NBAT-1 into the binary logistic regression equation, the regression value Y of each sample can be obtained, and the possible regression value Y is used as a diagnostic point to calculate the sensitivity and specificity, and the ROC curve is drawn accordingly. Figure 1), AUC is 0.921, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index can be diagnosed to distinguish the best cut-off between the Luminal A breast cancer non-metastasis group and the bone metastasis group. The value is 0.598 (the diagnostic threshold).

4. Verification to verify the accuracy of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 relative expression level in distinguishing Luminal A breast cancer from non-metastasis and Luminal A breast cancer bone metastasis

In the validation set, the relative expression levels of each sample lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 were substituted into the above regression model, and the regression values Y, Y of each sample were higher than the diagnostic threshold of 0.598, and the Luminal A breast cancer bone metastasis was predicted. A prediction below the diagnostic threshold of 0.598 is Luminal type A breast cancer without metastasis, with an accuracy of 98.2% (108/110), as shown in Figure 2.

Example 2: Luminal B breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

Test set and validation set of Luminal B type breast cancer non-metastasis group and Luminal B type breast cancer bone metastasis group.

2. RNA extraction and qRT-PCR

Total RNA was extracted from serum samples using Trizol reagent (Invitrogen, Shanghai, China). The concentration and purity of total RNA were measured at 260 nm using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific), and electrophoretic detection revealed that the purified RNA was of good quality and then subjected to subsequent operations. Total RNA was converted to cDNA using a reverse transcription kit (Takara, Dalian, China). Fluorescence quantitative PCR (Takara, Dalian, China) was performed using the SYBR Green method, and data collection was performed using an ABI Prism 7000 fluorescent quantitative PCR system (Agilent Technologies). The primer was synthesized by Beauchamps (Shanghai, China), lncRNA XLOC_004122 Primer sequence: upstream 5'-CTGGCAGGAACACCGGGTACTT-3', downstream 5'-TGACTTTTACTTAGGAGCCACTTCTTG-3'; lncRNA Linc00467 Primer sequence: upstream 5'-GCCTG GTTGTTCAGCACCTTCG-3', downstream 5'-TCGGATCGGTGCTGGTTTTGGT-3'; lncRNA Al049452 Primer sequence: upstream 5'-CAGTTAAACCCACAGGTGGTAGCATGAC-3', downstream 5'-TAGTGGGAAAA CCTAGTTTCCGACAGTT-3'; GAPDH primer sequence: upstream 5'-CGCTCTCTGCTCCTCCTGTTC-3', downstream 5'-ATCCGTTGACTCCGACCTTCAC -3'. The relative expression of lncRNA XLOC_004122, lncRNA Linc00467 lncRNA Al049452 and an average value of 3 measurements ² -ΔΔCt calculation method.

3, statistical processing

Second, the experimental results

1. Relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in Luminal B breast cancer without metastasis and bone metastasis

In the test set, the relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 were determined for each sample. Compared with the Luminal B breast cancer non-metastasis group, the relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 were significantly up-regulated in the Luminal B breast cancer bone metastasis group. The relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in the bone metastasis group. They were (2.2±0.4) times, (2.7±0.3) times, and (2.3±0.3) times the relative expression levels of the untransferred group, respectively.

2, lncRNA XLOC_004122, lncRNA Linc00467 or lncRNA Al049452 relative expression level alone for the diagnosis of Luminal B type breast cancer without metastasis and Luminal B type breast cancer bone transfer ROC curve analysis

The relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 or lncRNA Al049452 were mapped in SPSS 19.0 alone to diagnose the ROC curves of Luminal B breast cancer unmetastatic and Luminal B breast cancer bone metastases, AUC were 0.687, 0.744, 0.706, respectively. Lower or medium accuracy.

3. Construction of a combined diagnostic model for relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 and ROC curve analysis for differential diagnosis of Luminal B breast cancer without metastasis and Luminal B breast cancer with bone metastasis

The relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in the test set samples were used as independent variables (X ₁ = lncRNA Linc00467 relative expression level, X ₂ = lncRNA Al049452 relative expression level, X ₃ = lncRNA XLOC_004122 relative expression level), As a dependent variable, the lncRNA XLOC_004122, lncRNA Linc00467, and lncRNA Al049452 were used in Luminal B breast cancer non-metastasis and Luminal B breast cancer bone metastasis samples by group (ie, whether the sample belongs to the bone metastasis group or the non-metastasis group according to the gold standard). The relative expression level was subjected to binary logistic regression to obtain a binary logistic regression equation: Y=-2.241+1.883X ₁ +2.275X ₂ +1.975X ₃ ; and the relative expression of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in each sample. Substituting the binary logistic regression equation horizontally, the regression value Y of each sample can be obtained, and the possible regression value Y is used as a diagnosis point, and the sensitivity and specificity are calculated, and the ROC curve is drawn accordingly (as shown in FIG. 3), and the AUC is 0.935, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index can be diagnosed to distinguish the best cut-off between the Luminal B type breast cancer non-metastasis group and the bone metastasis group. The value is 0.607 (the diagnostic threshold).

4. Verification to verify the accuracy of the combined expression of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in the differential diagnosis of Luminal B breast cancer without metastasis and Luminal B breast cancer.

In the validation set, the relative expression levels of each sample lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 were substituted into the above regression model, and the regression values Y, Y of each sample were higher than the diagnostic threshold of 0.607. The prediction was Luminal B type breast cancer bone metastasis, lower than The diagnostic threshold of 0.607 was predicted to be Luminal B breast cancer without metastasis with an accuracy of 95.2% (60/63), as shown in Figure 4.

Example 3: Her-2 overexpressing breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

Test set and validation set of Her-2 overexpressing breast cancer non-metastasis group and Her-2 overexpressing breast cancer bone metastasis group.

2. RNA extraction and qRT-PCR

Total RNA was extracted from serum samples using Trizol reagent (Invitrogen, Shanghai, China). The concentration and purity of total RNA were measured at 260 nm using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific), and electrophoretic detection revealed that the purified RNA was of good quality and then subjected to subsequent operations. Total RNA was converted to cDNA using a reverse transcription kit (Takara, Dalian, China). Fluorescence quantitative PCR (Takara, Dalian, China) was performed using the SYBR Green method, and data collection was performed using an ABI Prism 7000 fluorescent quantitative PCR system (Agilent Technologies). The primer was synthesized by Beauchamps (Shanghai, China), lncRNA AK043773 primer sequence: upstream 5'-GTGACGCCAGGGATGGCATTA-3', downstream 5'-CAG AGCCTTGCATTGGTCAGT-3'; lncRNA EXOC7 primer sequence: upstream 5'-GAGTCTGGGATCAGAGA GCAAAGG-3', Downstream 5'-GGTACTGTAGAAAGGCCCCGTAGG-3'; GAPDH primer sequence: upstream 5'-C GCTCTCTGCTCCTCCTGTTC-3', downstream 5'-ATCCGTTGACTCCGACCTTCAC-3'. Relative expression lncRNA AK043773 lncRNA EXOC7 and an average value of 3 measurements ² -ΔΔCt calculation method.

3, statistical processing

Second, the experimental results

1. Relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in Her-2 overexpressing breast cancer non-metastasis group and bone metastasis group

In the test set, the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 were determined for each sample. Compared with the Her-2 overexpressing breast cancer non-metastasis group, the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in the Her-2 overexpressing breast cancer bone metastasis group were significantly up-regulated, and the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in the bone metastasis group. They were (3.3 ± 0.5) times and (2.6 ± 0.3) times of the untransferred group, respectively.

2. The relative expression level of lncRNA AK043773 or lncRNA EXOC7 was used to diagnose the ROC curve of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer.

The relative expression levels of lncRNA AK043773 or lncRNA EXOC7 were mapped in SPSS 19.0 alone to diagnose the ROC curves of bone metastases in Her-2 overexpressing breast cancer and meta-2 overexpressing breast cancer, with AUC of 0.762 and 0.717, respectively. Has moderate accuracy.

3. Construction of a combined diagnostic model for relative expression levels of lncRNA AK043773 and lncRNA EXOC7 and ROC curve analysis for differential diagnosis of bone metastases in Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer

The relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in the test set samples were used as independent variables (X ₁ = lncRNA AK043773 relative expression level, X ₂ = lncRNA EXOC7 relative expression level), in groups (ie, the sample belongs to bone according to the gold standard) Binary Logistic Regression of Relative Expression Levels of lncRNA AK043773 and lncRNA EXOC7 in Her-2 Overexpressing Breast Cancer Unmetastatic and Her-2 Overexpressing Breast Cancer Bone Metastasis Samples as a Dependent Variable , the binary logistic regression equation is obtained: Y=-2.918+2.618X ₁ +2.115X ₂ ; and the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in each sample are substituted into the binary logistic regression equation to obtain the regression of each sample. The value Y, with the possible regression value Y as the diagnostic point, calculates the sensitivity and specificity, and draws the ROC curve (shown in Figure 5) accordingly, with an AUC of 0.939, with high accuracy. Further, according to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon index can be diagnosed to distinguish the Her-2 overexpressing breast cancer from the non-metastasis group and the bone metastasis group. The cut-off value is 0.495 (ie, the diagnostic threshold).

4. Verification to verify the accuracy of lncRNA AK043773 and lncRNA EXOC7 relative expression level in the diagnosis of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer bone metastasis

In the validation set, the relative expression levels of each sample lncRNA AK043773 and EXOC7 were substituted into the above regression model, and the regression values Y, Y of each sample were higher than the diagnostic threshold of 0.495. The prediction was Her-2 overexpressing breast cancer bone metastasis, lower than diagnosis. The threshold of 0.495 was predicted to be non-metastatic in Her-2 overexpressing breast cancer with an accuracy of 91.1% (51/56), as shown in Figure 6.

Example 4: Triple negative breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

Test set and validation set of triple-negative breast cancer non-metastasis group, triple-negative breast cancer bone metastasis group.

2. RNA extraction and qRT-PCR

Total RNA was extracted from serum samples using Trizol reagent (Invitrogen, Shanghai, China). The concentration and purity of total RNA were measured at 260 nm using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific), and electrophoretic detection revealed that the purified RNA was of good quality and then subjected to subsequent operations. Total RNA was converted to cDNA using a reverse transcription kit (Takara, Dalian, China). Fluorescence quantitative PCR (Takara, Dalian, China) was performed using the SYBR Green method, and data collection was performed using an ABI Prism 7000 real-time PCR system (Agilent Technologies). The primer was synthesized by Beauchamps (Shanghai, China), lncRNA Lnc01089 Primer sequence: upstream 5'-TCGCTGGGTTGCTCTGCTTC-3', downstream 5'-GTCAGGAGGTCACAGTCTTAGGG-3'; lncRNA HOTAIR primer sequence: upstream 5'-CGTGGAAAGATCCAAATGGGACCA-3', downstream 5 '-AGCCTAGGAATCAGCACGAAGCAAA-3'; GAPDH primer sequence: upstream 5'-CGCTCTCTGCTCCTCCTGTTC-3', downstream 5'-ATCCGTTGACTCCGACCTTCAC-3'. Relative expression lncRNA Lnc01089 lncRNA HOTAIR and an average value of 3 measurements ² -ΔΔCt calculation method.

3, statistical processing

Second, the experimental results

1. Relative expression levels of lncRNA Lnc01089 and HOTAIR in the triple-negative breast cancer non-metastasis group and bone metastasis group

In the test set, the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR were determined for each sample. Compared with the triple-negative breast cancer non-metastasis group, the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR were significantly up-regulated in the triple-negative breast cancer bone metastasis group. The relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in the bone metastasis group were untransferred group, respectively. (3.5 ± 0.6) times, (3.2 ± 0.5) times.

2, lncRNA Lnc01089 or lncRNA HOTAIR relative expression level alone for the diagnosis of three negative breast cancer without metastasis and triple negative breast cancer bone transfer ROC curve analysis

The relative expression levels of lncRNA Lnc01089 or lncRNA HOTAIR were mapped in SPSS 19.0 alone to diagnose the ROC curves of triple-negative breast cancer unmetastatic and triple-negative breast cancer bone metastases with AUC of 0.755 and 0.732, respectively. .

3. Construction of a combined diagnostic model for relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR and ROC curve analysis for differential diagnosis of triple-negative breast cancer without metastasis and triple-negative breast cancer with bone metastasis

The relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in the test set samples were used as independent variables (X ₁ = lncRNA Lnc01089 relative expression level, X ₂ = lncRNA HOTAIR relative expression level), in groups (ie, the sample belongs to bone according to the gold standard) Binary logistic regression was performed as a dependent variable for the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in triple-negative breast cancer unmetastatic and triple-negative breast cancer bone metastasis samples, resulting in binary logistic regression. Equation: Y=-2.537+2.793X ₁ +2.181X ₂ ; Substituting the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in each sample into the binary logistic regression equation, the regression value Y of each sample can be obtained, possibly The regression value Y is used as a diagnostic point to calculate the sensitivity and specificity, and the ROC curve is plotted accordingly (as shown in Fig. 7), and the AUC is 0.948, which has high accuracy. Further, according to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index can be diagnosed to distinguish the best cut of the triple-negative breast cancer non-metastasis group and the bone metastasis group. The off value is 0.633 (ie the diagnostic threshold).

4. Verification to verify the accuracy of lncRNA Lnc01089 and lncRNA HOTAIR relative expression levels in the diagnosis of triple-negative breast cancer without metastasis and triple-negative breast cancer bone metastasis

In the validation set, the relative expression levels of each sample lncRNA Lnc01089 and lncRNA HOTAIR were substituted into the above regression model, and the regression values Y, Y of each sample were higher than the diagnostic threshold of 0.633. The prediction was triple-negative breast cancer bone metastasis, which was lower than the diagnostic threshold of 0.633. The prediction was that triple-negative breast cancer did not metastasize with an accuracy of 92.9% (52/56), as shown in Figure 8.

In summary, the present inventors have found that serum lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 can be used in combination to diagnose the bone metastasis of Luminal A breast cancer, and the accuracy of the independent verification is more than 90%; serum lncRNA XLOC_004122 , lncRNA Linc00467 and lncRNA Al049452 can be used in combination to diagnose the bone metastasis of Luminal B breast cancer. The accuracy of the independent diagnosis is more than 90%. The serum lncRNA AK043773 and lncRNA EXOC7 can be used together for the diagnosis of Her-2. Whether the expression of breast cancer is bone metastasis, the accuracy of independent diagnosis in the independent verification predicts more than 90%; lncRNA Lnc01089 and lncRNA HOTAIR can be used together to diagnose the bone metastasis of triple-negative breast cancer, and the accuracy of the independent diagnosis is predictive. More than 90. The use of the above-mentioned serum lncRNA diagnosis indicates that the bone metastasis of different molecular subtypes of breast cancer is not only highly accurate, but also has low detection cost, non-invasiveness, convenience, and greatly reduces the pain and burden of the patient.

Part II: Methylation gene diagnostic composition and diagnostic method

The experimental sample is the same as the first part. Serum specimen collection: 5.0 mL of fasting venous blood of the patient was collected, and the serum was isolated after centrifugation (4000 r/min, 2860×g) for 7 min after natural coagulation, and stored at -80 ° C for detection of the target methylation gene in serum.

Example 5: Luminal type A breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

2, serum total DNA extraction

Serum DNA extraction was performed according to the DNA Blood Midi Kit instructions, using 0.8 mL of serum per sample. The purity of the extracted DNA was detected by an ultraviolet spectrophotometer, and the ratio of the absorbance A260/A280 was between 1.7 and 2.0 for subsequent operations. Calculate the DNA content and store at -70 °C for later use.

3. DNA sulfite modification and pyrosequencing detection

DNA sulfite modification:

1 μg of DNA was taken, and the genomic DNA was methylated according to the DNA Methylation-Goldkit instructions, and stored at -70 ° C until use. Polymerase chain reaction: PCR was used to amplify the methylation region of the PITX1 and AMOT gene promoters in the sample. The reaction system includes sulfite-treated template 2 μl, 10×PCR buffer, 0.25 U/μl Hot star Taq enzyme, 0.5 mmol/L dNTP, 1 μl of each of the upstream and downstream primers, and a total volume of 50 μl. Double distilled water was used as a blank control.

Pyrosequencing detection:

(1) 45 μl of PCR amplification products were separately taken into PSQ 96 Plate Low sample preparation plate A, and 45 μl of binding buffer and 8 μl of streptavidin-coated magnetic beads were added, and shaken at 43 ° C for 25 min. The magnetic beads bound to the PCR product were transferred to plate B of the denaturing buffer to sufficiently denature the double-stranded DNA. The magnetic beads bound to the single-stranded PCR product were transferred to a plate C of 150 μl of annealing buffer for 3 min vortexing.

(2) Sequencing primer hybridization: The magnetic beads bound to the single-stranded PCR product were transferred into 50 μl of refolding buffer, 10 μl of sequencing primer was added, and hybridization was carried out at 75 ° C for 7 min.

(3) Using the PSQ96 pyrosequencing instrument and sequencing reaction kit (PyroGoldReagents), the base frequencies of the methylation sites in the PITX1 and AMOT promoter regions were detected, respectively.

The PCR amplification primers and sequencing primers are as follows:

PITX1

Upstream 5’-GGAAGGTATTTAGTATAGGTGAGTTTGA-3’

Downstream 5'-AAACCTTAATATTCACTACACTTTATC-3’

Sequencing 5’-GTGTTTATTTTGGATTGTTTAATT-3’

AMOT

Upstream 5’-TGAGTTAATATGAAAGAAGATAGTA-3’

Downstream 5'-TGATCTCTACATCTCAACTAATATAC-3’

Sequencing 5’-GTAGGTTTATTTAGGTT-3’

Each methylation site was analyzed by using a pyrosequencing allele frequency analysis function. The methylation index of the promoter region of each gene was calculated according to the following formula, which reflects the degree of methylation of the promoter region of the gene:

4, statistical processing

The data was analyzed using SPSS 19.0. The measurement data were expressed as mean ± deviation. The t test was used. The count data was expressed as a percentage. The χ ² test was used. The difference was statistically significant at P < 0.05. The ROC curve was established and the area under the curve (areaunderthecurve, AUC) and 95 were calculated. % confidence interval. Logistic regression was used to screen the variables, and regression equations were established to generate a new set of variables Y. The ROC curve analysis was performed on the new variables and each individual indicator.

Second, the experimental results

1. Degree of methylation of methylated PITX1 and methylated AMOT in Luminal type A breast cancer without metastasis and bone metastasis

In the test set, the methylation index of methylated PITX1 and methylated AMOT in each sample was determined. Compared with the Luminal type A breast cancer non-metastasis group, the methylation index of methylated PITX1 and methylated AMOT was significantly increased in the Luminal type A breast cancer bone metastasis group. The bone metastasis group methylated PITX1 and methyl group. The methylation index of AMOT was (3.5±0.4) times and (3.3±0.5) times of the methylation index of the untransferred group, respectively.

2. The methylation index of methylated PITX1 and methylated AMOT was used to diagnose the ROC curve of Luminal A breast cancer without metastasis and Luminal A breast cancer.

The methylation index of methylated PITX1 and methylated AMOT was plotted in SPSS 19.0 alone to diagnose the ROC curve for the differential metastasis of Luminal A breast cancer and Luminal A breast cancer. The AUC was 0.715 and 0.707, respectively. Has moderate accuracy.

3. Construction of a combined diagnostic model for methylation index of methylated PITX1 and methylated AMOT and ROC curve analysis for differential diagnosis of Luminal A breast cancer without metastasis and Luminal A breast cancer bone metastasis

The methylation index of methylated PITX1 and methylated AMOT in the test set samples was used as an independent variable (X ₁ = methylation index of methylated PITX1, X ₂ = methylation index of methylated AMOT) As a dependent variable, whether the sample belongs to the bone metastasis group or the non-metastasis group according to the gold standard, methylation of PITX1 and methylated AMOT in Luminal type A breast cancer without metastasis and Luminal type A breast cancer bone metastasis The methylation index in the sample is subjected to binary logistic regression to obtain a binary logistic regression equation: Y=1/[1+EXP(1.499X ₁ +2.302X ₂ -0.258)];

Substituting the methylation index of methylated PITX1 and methylated AMOT in each sample into the binary logistic regression equation, the regression value Y of each sample can be obtained, and the possible regression value Y is used as a diagnostic point to calculate the sensitivity and Specificity, according to which the ROC curve is plotted (as shown in Figure 9), the AUC is 0.935, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index can be diagnosed to distinguish the best cut-off between the Luminal A breast cancer non-metastasis group and the bone metastasis group. The value is 0.238 (the diagnostic threshold).

4. Verification to verify the accuracy of methylation index of methylated PITX1 and methylated AMOT in the diagnosis of Luminal A breast cancer without metastasis and Luminal A breast cancer bone metastasis

In the validation set, the methylation index of each sample methylated PITX1 and methylated AMOT was substituted into the above regression model, and the regression value Y of each sample was obtained. Y is lower than the diagnostic threshold of 0.238. The prediction is Luminal type A breast cancer bone metastasis. The prediction above the diagnostic threshold of 0.238 is Luminal type A breast cancer without metastasis, with an accuracy of 95.5% (105/110), as shown in Figure 10.

Example 6: Luminal B breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

2, serum total DNA extraction

3. DNA sulfite modification and pyrosequencing detection

DNA sulfite modification:

1 μg of DNA was taken, and the genomic DNA was methylated according to the DNA Methylation-Goldkit instructions, and stored at -70 ° C until use. Polymerase chain reaction: The methylation region of the PTPN1 and SLIT2 gene promoters in the sample was amplified by PCR. The reaction system includes sulfite-treated template 2 μl, 10×PCR buffer, 0.25 U/μl Hot star Taq enzyme, 0.5 mmol/L dNTP, 1 μl of each of the upstream and downstream primers, and a total volume of 50 μl. Double distilled water was used as a blank control.

Pyrosequencing detection:

(3) The base frequencies of the methylation sites in the promoter regions of PTPN1 and SLIT2 were detected using a PSQ96 pyrosequencing instrument and a sequencing reaction kit (Pyro Gold Reagents), respectively.

The PCR amplification primers and sequencing primers are as follows:

PTPN1

Upstream 5’-AGCGGGTTAGAGGGTAGATGT-3’

Downstream 5'-TAGGTTTCTCCTCTCCCACATAT-3’

Sequencing 5'-TTTCCATTCATCCTAA-3'

SLIT2

Upstream 5'-TGAAGTTTTATTAGGTTGTGGAGGAGTA-3’

Downstream 5'-ATACCAAATATCCTATCCTTATCTTC-3’

Sequencing 5'-GTTTAAGGTTTATGATA-3'

4, statistical processing

Second, the experimental results

1. Degree of methylation of methylated PTPN1 and methylated SLIT2 in Luminal B breast cancer without metastasis and bone metastasis

In the test set, the methylation index of methylated PTPN1 and methylated SLIT2 in each sample was determined. Compared with the Luminal B breast cancer non-metastasis group, the methylation index of methylated PTPN1 and methylated SLIT2 was significantly increased in the Luminal B breast cancer bone metastasis group. The bone metastasis group methylated PTPN1 and methyl group. The methylation index of SLIT2 was (2.9±0.5) times and (3.4±0.5) times that of the untransformed group methylation index, respectively.

2. The methylation index of methylated PTPN1 and methylated SLIT2 was used to diagnose the ROC curve of Luminal B breast cancer without metastasis and Luminal B breast cancer.

The methylation index of methylated PTPN1 and methylated SLIT2 was mapped in SPSS 19.0 alone to diagnose the ROC curve of Luminal B breast cancer unmetastatic and Luminal B breast cancer bone metastases, with AUC of 0.723 and 0.741, respectively. Has moderate accuracy.

3. Construction of a combined diagnostic model for methylation index of methylated PTPN1 and methylated SLIT2 and ROC curve analysis for differential diagnosis of Luminal B breast cancer without metastasis and Luminal B breast cancer with bone metastasis

The methylation index of methylated PTPN1 and methylated SLIT2 in the test set samples was used as an independent variable (X ₁ = methylation index of methylated PTPN1, X ₂ = methylation index of methylated SLIT2) As a dependent variable, the group of methylated PTPN1 and methylated SLIT2 in Luminal B type breast cancer without metastasis and Luminal B type breast cancer bone metastasis The methylation index in the sample was subjected to binary logistic regression to obtain a binary logistic regression equation: Y=1/[1+EXP(2.016X ₁ +1.898X ₂ -0.455)];

Substituting the methylation index of methylated PTPN1 and methylated SLIT2 in each sample into the binary logistic regression equation, the regression value Y of each sample can be obtained, and the possible regression value Y is used as a diagnostic point to calculate the sensitivity and Specificity, according to which the ROC curve is plotted (as shown in Figure 11), the AUC is 0.942, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index can be diagnosed to distinguish the best cut-off between the Luminal B type breast cancer non-metastasis group and the bone metastasis group. The value is 0.310 (the diagnostic threshold).

4. Verification to verify the accuracy of methylation index of methylated PTPN1 and methylated SLIT2 in the diagnosis of Luminal B breast cancer without metastasis and Luminal B breast cancer bone metastasis

In the validation set, the methylation index of each sample methylated PTPN1 and methylated SLIT2 was substituted into the above regression model, and the regression value Y of each sample was obtained. Y is lower than the diagnostic threshold of 0.310. The prediction is Luminal B type breast cancer bone metastasis. The prediction above the diagnostic threshold of 0.310 was that Luminal B breast cancer was not metastatic, with an accuracy of 93.7% (59/63), as shown in Figure 12.

Example 7: Her-2 overexpressing breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

2, serum total DNA extraction

3. DNA sulfite modification and pyrosequencing detection

DNA sulfite modification:

1 μg of DNA was taken, and the genomic DNA was methylated according to the DNA Methylation-Goldkit instructions, and stored at -70 ° C until use. Polymerase chain reaction: PCR was used to amplify the methylation region of the MYLK2, EFEMP1 and SOSTDC1 gene promoters in the sample. The reaction system includes sulfite-treated template 2 μl, 10×PCRbuffer, 0.25 U/μl Hot star Taq enzyme, 0.5 mmol/L dNTP, 1 μl of each of the upstream and downstream primers, and a total volume of 50 μl.

Pyrosequencing detection:

(3) The base frequencies of methylation sites in the promoter regions of MYLK2, EFEMP1 and SOSTDC1 were detected using a PSQ96 pyrosequencing instrument and a sequencing reaction kit (Pyro Gold Reagents).

The PCR amplification primers and sequencing primers are as follows:

MYLK2

Upstream 5’-GAGGGAAAGGATATGGTTGATT-3’

Downstream 5’-AACTCCACTCCATTCTCCC-3’

Sequencing 5'-AGTAAGTTATTTATTTGTTATTTG-3’

EFEMP1

Upstream 5’-GGTTTAGGTGGGGAGTATGATAG-3’

Downstream 5'-ACCAACAACCCAACTTTAACATAACC-3’

Sequencing 5'-TAATGAGGGGTTGAG-3'

SOSTDC1

Upstream 5'-GTAAAGGAGAAAGTTTGGTATATGG-3’

Downstream 5'-CAAAACTATACAAAAGTATCTCTCTCAAT-3’

Sequencing 5’-ATAATTTAATTGTTAGAGTTGAATA-3’

4, statistical processing

Second, the experimental results

1. Methylation of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in Her-2 overexpressing breast cancer without metastasis and bone metastasis

In the test set, the methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in each sample was determined. Compared with the Her-2 overexpressing breast cancer non-metastasis group, the methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 was significantly increased in the Her-2 overexpressing breast cancer bone metastasis group. , (3.7±0.6) times, (2.6±0.4), (3.1±0.5) times of the methylation index of the untransferred group, respectively.

2. Methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 was used to diagnose ROC curve of bone metastasis of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer. analysis

The methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 was mapped in SPSS 19.0 alone to diagnose the differentiation of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer. The ROC curve, AUC, is 0.794, 0.688, 0.738, respectively, with low or medium accuracy.

3. Construction of a combined methylation index model for methylation of MYLK2, methylated EFEMP1 and methylated SOSTDC1 and its use in the diagnosis of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer ROC curve analysis of bone metastasis

The methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in the test sample was used as the independent variable (X ₁ = methylation index of methylated MYLK2, X ₂ = methylated EFEMP1) Methylation index, X ₃ = methylation index of methylated SOSTDC1), as a dependent variable (ie, whether the sample belongs to the bone metastasis group or the non-transfer group according to the gold standard), methylated MYLK2, methyl The binary logistic regression of the methylation index of EFEMP1 and methylated SOSTDC1 in Her-2 overexpressing breast cancer unmetastatic and Her-2 overexpressing breast cancer bone metastasis samples, and the binary logistic regression equation was obtained: Y =1/[1+EXP(1.342X ₁ +1.401X ₂ +1.345X ₃ -2.035)];

The methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in each sample was substituted into the binary logistic regression equation to obtain the regression value Y of each sample, and the possible regression value Y was used as a diagnosis. Point, calculate the sensitivity and specificity, and draw the ROC curve (as shown in Figure 13), with an AUC of 0.950, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon index can be diagnosed to distinguish the Her-2 overexpressing breast cancer from the non-metastasis group and the bone metastasis group. The cut-off value is 0.308 (diagnostic threshold).

4. Verification to verify the methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 combined with diagnosis to distinguish the bone metastasis of Her-2 overexpressing breast cancer without metastasis and Her-2 overexpressing breast cancer degree

In the validation set, the methylation index of each sample methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 was substituted into the above regression model to obtain the regression value Y of each sample, and the prediction of the sample below the diagnostic threshold of 0.308 was Her- 2 Over-expression breast cancer bone metastasis, higher than the diagnostic threshold of 0.308 predicted that Her-2 overexpressing breast cancer did not metastasize, the accuracy was 96.4% (54/56), as shown in Figure 14.

Example 8: Three-negative breast cancer bone metastasis

First, experimental samples and experimental methods

1. Experimental sample

2, serum total DNA extraction

3. DNA sulfite modification and pyrosequencing detection

DNA sulfite modification:

1 μg of DNA was taken, and the genomic DNA was methylated according to the DNA Methylation-Gold kit instructions, and stored at -70 ° C until use. Polymerase chain reaction: PCR was used to amplify the methylation region of the MYLK3 and SCARA5 gene promoters in the sample. The reaction system includes sulfite-treated template 2 μl, 10×PCR buffer, 0.25 U/μl Hot star Taq enzyme, 0.5 mmol/L dNTP, 1 μl of each of the upstream and downstream primers, and a total volume of 50 μl. Double distilled water was used as a blank control.

Pyrosequencing detection:

(3) Using the PSQ96 pyrosequencing instrument and the sequencing reaction kit (Pyro Gold Reagents), the base frequencies of the methylation sites in the promoter regions of MYLK3 and SCARA5 were detected, respectively.

The PCR amplification primers and sequencing primers are as follows:

MYLK3

Upstream 5’-TAGGGGAGGTTAAGAAAGTGTA-3’

Downstream 5'-AACTCCTTATCAATTCCTAACATACAAT-3’

Sequencing 5'-GGAGTAATGATGTAATGTGTAT-3'

SCARA5

Upstream 5’-AGGAATTAGGTAAGGTATGTTAGTA-3’

Downstream 5'-AAAACTCCAACCTATTCCAACCATACCTAC-3’

Sequencing 5'-GTTTTAAGTTTTGGTGTTTGATAT-3’

4, statistical processing

Second, the experimental results

1. Methylation of methylated MYLK3 and methylated SCARA5 in triple-negative breast cancer without metastasis and bone metastasis

In the test set, the methylation index of methylated MYLK3 and methylated SCARA5 in each sample was determined. Compared with the triple-negative breast cancer non-metastasis group, the methylation index of methylated MYLK3 and methylated SCARA5 was significantly increased in the triple-negative breast cancer bone metastasis group, and the bone metastasis group methylated MYLK3 and methyl group. The methylation index of SCARA5 was (2.1±0.3) times and (3.6±0.7) times that of the untransformed group methylation index, respectively.

2. The methylation index of methylated MYLK3 and methylated SCARA5 was used to diagnose the ROC curve of triple-negative breast cancer without metastasis and triple-negative breast cancer.

The methylation index of methylated MYLK3 and methylated SCARA5 was mapped in SPSS 19.0 alone to diagnose the ROC curve of three-negative breast cancer unmetastatic and triple-negative breast cancer bone metastases, AUC were 0.644, 0.809, respectively. Has low or medium accuracy.

3. Construction of a combined diagnostic model for methylation index of methylated MYLK3 and methylated SCARA5 and ROC curve analysis for diagnosis of bone metastases in triple-negative breast cancer without metastasis and triple-negative breast cancer

The methylation index of methylated MYLK3 and methylated SCARA5 in the test sample was used as the independent variable (X ₁ = methylation index of methylated MYLK3, X ₂ = methylation index of methylated SCARA5) As a dependent variable, the methylation of MYLK3 and methylated SCARA5 in triple-negative breast cancer without metastasis and triple-negative breast cancer bone metastasis The methylation index in the sample was subjected to binary logistic regression to obtain a binary logistic regression equation: Y=1/[1+EXP(1.775X ₁ +1.236X ₂ -0.398)];

Substituting the methylation index of methylated MYLK3 and methylated SCARA5 in each sample into the binary logistic regression equation, the regression value Y of each sample can be obtained, and the possible regression value Y is used as a diagnostic point to calculate the sensitivity and Specificity, according to which the ROC curve is plotted (as shown in Figure 15), the AUC is 0.954, with high accuracy. According to the coordinates of the ROC curve, the Verdon index = specificity + sensitivity-1, and the corresponding Y value of the maximum value of the Verdon's index is the best cut-off for the diagnosis of the triple-negative breast cancer non-metastasis group and the bone metastasis group. The value is 0.366 (the diagnostic threshold).

4. Verification to verify the accuracy of methylation index of methylated MYLK3 and methylated SCARA5 in the diagnosis of triple-negative breast cancer without metastasis and triple-negative breast cancer

In the validation set, the methylation index of each sample methylated MYLK3 and methylated SCARA5 was substituted into the above regression model, and the regression value Y of each sample was obtained. Y is lower than the diagnostic threshold of 0.366 and is predicted to be triple negative breast cancer bone metastasis. The prediction above the diagnostic threshold of 0.366 was that triple-negative breast cancer did not metastasize with an accuracy of 94.6% (53/56), as shown in Figure 16.

In summary, the present inventors have found that serum methylated PITX1 and methylated AMOT can be used in combination to diagnose the bone metastasis of Luminal A breast cancer, and the accuracy of the independent verification is more than 90%; serum methylation PTPN1 and methylated SLIT2 can be used in combination to diagnose the bone metastasis of Luminal B breast cancer. The independent diagnosis has a predictive accuracy of more than 90%. Serum methylation of MYLK2, methylated EFEMP1 and methylated SOSTDC1 can be Combined use in the diagnosis of bone metastases in Her-2 overexpressing breast cancer, the accuracy of independent diagnosis in the independent validation predicts more than 90%; serum methylation of MYLK3 and methylated SCARA5 can be used in combination to predict the diagnosis of triple negative mammary gland Whether the cancer is bone metastasis or not, the accuracy of the independent verification indicates that the accuracy rate is over 90%. The use of the above-mentioned serum methylation gene diagnosis indicates that the bone metastasis of different molecular subtypes of breast cancer is not only highly accurate, but also has low detection cost, non-invasiveness, convenience, and greatly reduces the pain and burden of the patient.

The above embodiments are intended to specifically describe the substantial content of the present invention, but those skilled in the art should understand that the scope of the present invention should not be limited to the specific embodiments.

Claims

A lncRNA diagnostic composition for diagnosing bone metastasis of Luminal type A breast cancer, characterized by: lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1.
A method for diagnosing bone metastasis of Luminal type A breast cancer, comprising the steps of:

Step S1, collecting fasting venous blood of Luminal type A breast cancer patient, and separating the serum by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA XLOC_004122, lncRNA SUMO1P3 and lncRNA NBAT-1 in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X 3 , X 1 , X 2 ;

Step S3, substituting X 3 , X 1 , and X 2 values into a binary logistic regression equation Y=-2.577+2.045X 1 +1.956X 2 +1.676X 3 to obtain a Y value, and a Y value greater than 0.598 indicates that the breast cancer patient develops bone. Metastasis, less than 0.598, indicates that no bone metastasis has occurred.
A lncRNA diagnostic composition for diagnosing bone metastasis of Luminal type B breast cancer, characterized by: lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452.
A method for diagnosing bone metastasis of Luminal B type breast cancer, comprising: the following steps:

Step S1, collecting fasting venous blood of Luminal B type breast cancer patient, and separating the serum by centrifugation after natural coagulation;

Step S2, extracting total serum RNA, and determining the relative expression levels of lncRNA XLOC_004122, lncRNA Linc00467 and lncRNA Al049452 in total RNA relative to the internal reference GAPDH by qRT-PCR, represented by X 3 , X 1 , X 2 ;

Step S3, substituting X 3 , X 1 , and X 2 values into a binary logistic regression equation Y=Y=-2.241+1.883X 1 +2.275X 2 +1.975X 3 to obtain a Y value, and the Y value is greater than 0.607, indicating that the breast cancer patient Bone metastasis occurred, less than 0.607 indicates no bone metastasis.
A diagnostic composition for predicting Her-2 overexpressing breast cancer bone metastasis lncRNA characterized by: lncRNA AK043773 and lncRNA EXOC7.
A method for diagnosing bone metastasis of Her-2 overexpressing breast cancer, comprising the steps of:

In step S1, the fasting venous blood of the Her-2 overexpressing breast cancer patient is collected, and the serum is separated by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA AK043773 and lncRNA EXOC7 in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X 1 and X 2 ;

Step S3, substituting X 1 and X 2 values into a binary logistic regression equation Y=-2.918+2.618X 1 +2.115X 2 to obtain a Y value, and a Y value greater than 0.495 indicates that the breast cancer patient has bone metastasis, and less than 0.495 indicates that no occurrence occurs. Bone metastasis.
A diagnostic composition for predicting triple-negative breast cancer bone metastasis lncRNA, characterized by: lncRNA Lnc01089 and lncRNA HOTAIR.
A method for diagnosing bone metastasis of a triple negative breast cancer, comprising the steps of:

Step S1, collecting fasting venous blood of a triple-negative breast cancer patient, and separating the serum by centrifugation after natural coagulation;

In step S2, serum total RNA is extracted, and the relative expression levels of lncRNA Lnc01089 and lncRNA HOTAIR in total RNA relative to the internal reference GAPDH are determined by qRT-PCR, and are sequentially expressed by X 1 and X 2 ;

Step S3, substituting X 1 and X 2 values into a binary logistic regression equation Y=-2.537+2.793X 1 +2.181X 2 to obtain a Y value, and a Y value greater than 0.633 indicates that the breast cancer patient has bone metastasis, and less than 0.633 indicates that no occurrence occurs. Bone metastasis.
A methylation gene diagnostic composition for diagnosing bone metastasis of Luminal type A breast cancer, characterized by comprising methylated PITX1 and methylated AMOT.
A method for diagnosing bone metastasis of Luminal type A breast cancer, comprising the steps of:

Step S1, collecting fasting venous blood of Luminal type A breast cancer patient, and separating the serum by centrifugation after natural coagulation;

Step S2, extracting total serum DNA, and determining methylation index of methylated PITX1 and methylated AMOT in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X 1 and X 2 ;

Step S3, substituting the values of X 1 and X 2 into the binary logistic regression equation Y=1/[1+EXP(1.499X 1 +2.302X 2 -0.258)] to obtain a Y value, and the Y value is less than 0.238, indicating that the breast cancer patient occurs. Bone metastasis, greater than 0.238, indicates no bone metastasis.
A methylation gene diagnostic composition for diagnosing bone metastasis of Luminal type B breast cancer, characterized by comprising methylated PTPN1 and methylated SLIT2.
A method for diagnosing bone metastasis of Luminal B type breast cancer, comprising: the following steps:

Step S1, collecting fasting venous blood of Luminal B type breast cancer patient, and separating the serum by centrifugation after natural coagulation;

Step S2, extracting total serum DNA, and determining methylation index of methylated PTPN1 and methylated SLIT2 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X 1 and X 2 ;

Step S3, substituting the values of X 1 and X 2 into the binary logistic regression equation Y=1/[1+EXP(2.016X 1 +1.898X 2 -0.455)] to obtain a Y value, and the Y value is less than 0.310, indicating that the breast cancer patient occurs Bone metastasis, greater than 0.310, indicates no bone metastasis.
A diagnostic composition for predicting Her-2 overexpressing breast cancer bone metastasis methylation gene characterized by comprising methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1.
A method for diagnosing bone metastasis of Her-2 overexpressing breast cancer, comprising the steps of:

In step S1, the fasting venous blood of the Her-2 overexpressing breast cancer patient is collected, and the serum is separated by centrifugation after natural coagulation;

Step S2, extracting total serum DNA, and determining methylation index of methylated MYLK2, methylated EFEMP1 and methylated SOSTDC1 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, in turn X 1 , X 2 , X 3 ;

Step S3, substituting X 1 , X 2 , and X 3 into the equation Y=1/[1+EXP(1.342X 1 +1.401X 2 +1.345X 3 -2.035)] to obtain a Y value, and the Y value is less than 0.308, indicating that the breast cancer Bone metastasis occurred in the patient, and greater than 0.308 predicted no bone metastasis.
A diagnostic composition for diagnosing a triple-negative breast cancer bone metastasis methylation gene characterized by comprising methylated MYLK3 and methylated SCARA5.
A method for diagnosing bone metastasis of a triple negative breast cancer, comprising the steps of:

Step S1, collecting fasting venous blood of a triple-negative breast cancer patient, and separating the serum by centrifugation after natural coagulation;

Step S2, extracting total serum DNA, and determining methylation index of methylated MYLK3 and methylated SCARA5 in total DNA by PCR amplification, DNA sulfite modification and pyrosequencing, and sequentially expressed by X 1 and X 2 ;

Step S3, substituting the values of X 1 and X 2 into the binary logistic regression equation Y=1/[1+EXP(1.775X 1 +1.236X 2 -0.398)] to obtain a Y value, and the Y value is less than 0.366, indicating that the breast cancer patient occurs. Bone metastasis, greater than 0.366, indicates no bone metastasis.