A protein composition for predicting the prognosis of metastatic hormone-sensitive prostate cancer and use thereof

Abstract

The application provides a protein composition for predicting the prognosis effect of metastatic hormone-sensitive prostate cancer and application thereof, and belongs to the technical field of prognosis evaluation.The protein composition for predicting the prognosis effect of metastatic hormone-sensitive prostate cancer comprises protein molecules for evaluating drug resistance of a patient and protein molecules for evaluating sensitivity of the patient; the protein molecules for evaluating drug resistance of the patient comprise MCM4, NT5E, LTBP4, IAH1, HP, DOCK2 and EEF1A2; and the protein molecules for evaluating sensitivity of the patient comprise TFF3, FABP5 and LAMP2.The application constructs a molecular typing of mHSPC based on the above protein composition, evaluates the sensitivity of a mHSPC patient to ADT treatment in advance, and thus realizes precise treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of prognosis evaluation, and particularly relates to a protein composition for predicting the prognosis effect of metastatic hormone-sensitive prostate cancer and application thereof. BACKGROUND

[0002] Prostate cancer is the most common malignant tumor in elderly men. Prostate cancer is also the second leading cause of death from malignant tumors in male patients (accounting for 11%). Since Huggins et al. first confirmed in 1941 that the surgical castration method of bilateral testicular resection and estrogen therapy can inhibit the progression of prostate cancer, androgen deprivation therapy (ADT) has become the gold standard for the treatment of advanced prostate cancer. At the initial stage of ADT treatment, most patients are sensitive to the treatment, which is called hormone-sensitive prostate cancer (HSPC). However, after experiencing the initial 18-20 months of ADT sensitivity, most prostate cancer patients gradually develop drug resistance, and the tumor can still grow and metastasize in the case of extremely low androgen levels in the body, which is called castration-resistant prostate cancer (CRPC). At present, the mechanism of prostate cancer resistance still needs further research. In addition, due to individual differences of patients and heterogeneity of tumors, the sensitivity of different prostate cancer patients to ADT treatment also has great differences. This also causes different prostate cancer patients to have different benefits although they receive the same ADT treatment. At present, the focus and difficulty of the treatment of advanced prostate cancer is the lack of an effective prostate cancer molecular typing that can be used to predict the prognosis of patients, so as to screen out the high-benefit patient population of ADT in advance, and give more effective and more targeted precision treatment to the low-benefit population of ADT. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a protein composition for predicting the prognosis effect of metastatic hormone-sensitive prostate cancer, which can be used to perform HSPC typing according to the expression of the protein composition, so as to predict the prognosis effect of patients.

[0004] The present application provides a protein composition for predicting the prognosis effect of metastatic hormone-sensitive prostate cancer, which comprises protein molecules for evaluating the drug resistance of patients and protein molecules for evaluating the sensitivity of patients.

[0005] The protein molecules for evaluating the drug resistance of patients comprise MCM4, NT5E, LTBP4, IAH1, HP, DOCK2 and EEF1A2.

[0006] The protein molecules for evaluating the sensitivity of the patient include TFF3, FABP5 and LAMP2.

[0007] The application provides application of a reagent for detecting expression of the protein composition in construction of a molecular typing model for predicting sensitivity of a hormone-sensitive prostate cancer patient to androgen deprivation therapy.

[0008] Preferably, the reagent comprises at least one of the following: a reagent for protein immunoblotting detection, a reagent for immunohistochemical detection and a reagent for protein in-situ detection.

[0009] Preferably, when the reagent is the reagent for immunohistochemical detection, the method for constructing the typing model for predicting the prognostic effect of the hormone-sensitive prostate cancer comprises the following steps:

[0010] detecting the staining intensity and the positive rate of each protein molecule in the protein composition in the section sample of the hormone-sensitive prostate cancer patient, and calculating the final score of each protein molecule according to Formula I;

[0011] Final score = Staining intensity x Positive rate Formula I

[0012] the final score of each protein molecule is substituted into a nomogram typing calculation model to calculate a drug resistance score and a sensitivity score respectively, and an ADT score is calculated according to Formula II;

[0013] ADT score = Drug resistance score - Sensitivity score Formula II

[0014] molecular typing of the patient is performed according to the ADT score:

[0015] when the ADT score is <-1.5, the patient is of the ADT-sensitive type;

[0016] when the ADT score is >0.5, the patient is of the ADT-resistant type;

[0017] when the ADT score is -1.5-0.5, the patient is of the ADT-intermediate type.

[0018] Preferably, the drug resistance score is calculated according to Formula III;

[0019] Drug resistance score = -9.32942 + 1.74828 x Final score of MCM4 + 1.70248 x Final score of LTBP4 + 2.78289 x Final score of NT5E + 1.31930 x Final score of IAH1 + 3.65883 x Final score of DOCK2 - 0.11077 x Final score of HP - 1.71868 x Final score of EEF1A2 Formula III.

[0020] Preferably, the sensitivity score is calculated according to Formula IV;

[0021] The sensitivity score = -0.00090-0.22520 x TFF3 final score-0.68322 x FABP5 final score+0.55510 x LAMP2 final score Formula IV.

[0022] Preferably, the staining intensity is divided into four levels: no staining is 0 points, weak staining is 1 point, moderate staining is 2 points, and strong staining is 3 points.

[0023] The staining intensity is distinguished by the gray value determined by the IHC_Profiler function in the Image J software, the gray value of no staining is [0-60]; the gray value of weak staining is (120-180]; the gray value of moderate staining is (60-120]; and the gray value of strong staining is [0-60].

[0024] Preferably, the positive rate is divided into five levels: no positive cells is 0 points, <10% positive rate is 1 point, [10%~50%] interval positive rate is 2 points, (50%~70%] interval positive rate is 3 points, and >70% positive rate is 4 points.

[0025] Preferably, the positive rate is the percentage of the number of positive cells to the total number of cells.

[0026] The present application provides a protein composition for predicting the prognosis of metastatic hormone-sensitive prostate cancer, which comprises protein molecules for evaluating patient drug resistance and protein molecules for evaluating patient sensitivity; the protein molecules for evaluating patient drug resistance comprise MCM4, NT5E, LTBP4, IAH1, HP, DOCK2 and EEF1A2; and the protein molecules for evaluating patient sensitivity comprise TFF3, FABP5 and LAMP2. The present application classifies patients with confirmed advanced prostate cancer and receiving ADT treatment according to the sensitivity to ADT treatment into ADT sensitive HSPC, ADT resistant HSPC and ADT intermediate sensitive HSPC, further quantitatively detects proteomics and verifies differential proteins, and screens the above-mentioned 10 protein molecules protected by the present application. It is found by drawing the ROC curve that the AUC of the ROC curve of the drug resistance score is 0.92, and the AUC of the sensitivity score is 0.71. It can be seen that the protein composition provided by the present application has high sensitivity and specificity, and can be used for predicting or evaluating the effect of metastatic hormone-sensitive prostate cancer after ADT treatment. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Survival curves of three types of mHSPC patients;

[0028] Figure 2Hot map of proteomics results for three subtypes of mHSPC patients;

[0029] Figure 3 Nomogram of mHSPC molecular subtypes and its AUC curve, wherein A is nomogram of mHSPC drug resistance score; B is AUC curve of drug resistance score nomogram; C is decision curve analysis (DCA) of drug resistance score nomogram; D is nomogram of mHSPC sensitivity score; E is AUC curve of sensitivity score nomogram; F is decision curve analysis (DCA) of sensitivity score nomogram;

[0030] Figure 4 Immunohistochemical results of three subtypes of mHSPC patients, wherein A is 7 protein molecules of drug resistance score of three subtypes of mHSPC patients: MCM4, NT5E, LTBP4, IAH1, HP, DOCK2, EEF1A2; B is 3 protein molecules of sensitivity score of three subtypes of mHSPC patients: TFF3, FABP5, LAMP2. DETAILED DESCRIPTION

[0031] The present application provides a protein composition for predicting the prognosis of metastatic hormone-sensitive prostate cancer, comprising protein molecules for evaluating patient drug resistance and protein molecules for evaluating patient sensitivity; the protein molecules for evaluating patient drug resistance comprise MCM4, NT5E, LTBP4, IAH1, HP, DOCK2 and EEF1A2; the protein molecules for evaluating patient sensitivity comprise TFF3, FABP5 and LAMP2.

[0032] In the present application, the screening method of the protein composition preferably comprises the following steps:

[0033] Patients with advanced prostate cancer and receiving ADT treatment are divided into 3 types according to the sensitivity to ADT, and quantitative proteomics detection is continued for patients of the 3 types, 30 proteins with the largest difference are screened and verified, and 10 proteins are screened according to the analysis results of bioinformatics technology.

[0034] In the present application, the method for screening 10 proteins according to the analysis results of bioinformatics technology is to select 10 proteins with the highest sensitivity and specificity through nomogram. Based on the screened 10 proteins, drug resistance score and sensitivity score are calculated respectively, and ROC curves are drawn respectively, the AUC of the ROC curve of the drug resistance score is 0.92, and the AUC of the sensitivity score is 0.71. This shows that the 10 proteins screened in the present application have high sensitivity and specificity at the same time.

[0035] The application provides application of a reagent for detecting expression of the protein composition in construction of a molecular typing model for predicting sensitivity of a hormone-sensitive prostate cancer patient to androgen deprivation therapy.

[0036] In the application, the reagent preferably comprises at least one of the following: a reagent for protein immunoblotting detection, a reagent for immunohistochemical detection, and a reagent for protein in situ detection.

[0037] In the application, when the reagent is preferably a reagent for immunohistochemical detection, the method for constructing the typing model for predicting the prognostic effect of hormone-sensitive prostate cancer comprises the following steps:

[0038] The staining intensity and positive rate of each protein molecule in the protein composition in the section sample of the hormone-sensitive prostate cancer patient are detected, and the final score of each protein molecule is calculated according to formula I;

[0039] Final score = Staining intensity x Positive rate Formula I

[0040] The final score of each protein molecule is substituted into a typing calculation model nomogram to calculate a drug resistance score and a sensitivity score, respectively, and an ADT score is calculated according to formula II;

[0041] ADT score = Drug resistance score - Sensitivity score Formula II

[0042] The patient is molecularly typed according to the ADT score:

[0043] When the ADT score is <-1.5, the patient is of an ADT-sensitive type;

[0044] When the ADT score is >0.5, the patient is of an ADT-resistant type;

[0045] When the ADT score is -1.5-0.5, the patient is of an ADT intermediate type.

[0046] The method for immunohistochemically detecting the section sample of the hormone-sensitive prostate cancer patient in the application is not particularly limited, and a detection method well known in the art can be used. The reagent for immunohistochemical detection is commercially available. In the embodiments of the application, the specific detection method of immunohistochemistry is as follows: FFPE tissue specimens are cut into 5 μm, deparaffinized in xylene, and dehydrated through graded ethanol solutions; after heat mediation, sodium citrate antigen repair (10 mM sodium citrate, 0.05% Tween 20, pH = 6), the slide is coated with the purchased antibody of the target protein and the HRP kit and DAB substrate kit; the section is stained with hematoxylin and dehydrated through graded alcohol and xylene; a confocal microscope is used for examination and image processing, and the immunohistochemical section is observed under a light microscope, and the immunochromatographic intensity and positive cell density are used as quantitative scoring standards for interpretation.

[0047] In the present application, the staining intensity is preferably divided into four levels: no staining is 0 points, weak staining is 1 point, moderate staining is 2 points, and strong staining is 3 points. The staining intensity is distinguished by the gray value determined by the IHC_Profiler function in the Image J software, the gray value of no staining is [180-236]; the gray value of weak staining is [120-180]; the gray value of moderate staining is [60-120]; and the gray value of strong staining is [0-60]. The positive rate is preferably divided into five levels: no positive cells is 0 points, <10% positive rate is 1 point, [10%-50%] interval positive rate is 2 points, (50%-70%] interval positive rate is 3 points, and >70% positive rate is 4 points. The calculation method of the positive rate is the percentage of the number of positive cells to the total number of cells.

[0048] In the present application, the drug resistance score is preferably calculated according to formula III;

[0049] Drug resistance score = -9.32942 + 1.74828 x MCM4 final score + 1.70248 x LTBP4 final score + 2.78289 x NT5E final score + 1.31930 x IAH1 final score + 3.65883 x DOCK2 final score - 0.11077 x HP final score - 1.71868 x EEF1A2 final score Formula III.

[0050] In the present application, the sensitivity score is preferably calculated according to formula IV;

[0051] Sensitivity score = -0.00090 - 0.22520 x TFF3 final score - 0.68322 x FABP5 final score + 0.55510 x LAMP2 final score Formula IV.

[0052] In the present application, by studying the survival curves of patients with different molecular subtypes, it is found that the survival period of ADT-sensitive patients is longer, and the survival rate is higher than 50% at 150 months, the survival period of ADT-intermediate patients is shorter than that of ADT-sensitive patients, but longer than that of ADT-resistant patients, and the survival rate is about 50% at 50 months, and the survival period of ADT-resistant patients is the shortest. Therefore, the molecular typing model of mHSPC constructed by the present application can be used to evaluate the sensitivity of mHSPC patients to ADT treatment before the patients start treatment, and then screen for high-benefit patient population treated by ADT, and seek more precise treatment for low-benefit patient population treated by ADT.

[0053] The protein composition for predicting prognosis evaluation of metastatic hormone-sensitive prostate cancer and application thereof provided by the present application will be described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0054] Example 1

[0055] A screening method of a protein composition for predicting prognosis evaluation of metastatic hormone-sensitive prostate cancer

[0056] The case data of all patients diagnosed as advanced prostate cancer and treated by ADT in the past 10 years in the Second Hospital of Tianjin Medical University were collected, and a total of 527 cases were collected. The criteria for CRPC were as follows: the patient continuously received ADT treatment, and continuously had 3 or more than 3 serum PSA values increased, with an interval of ≥2 weeks, and the last value was >2.0 ng / mL, 2 times were greater than 50% of the minimum value or there were clear new lesions: bone scan showed more than 2 new bone lesions or CT / MR showed soft tissue progression. The time from the beginning of endocrine drug ADT treatment to the progression of CRPC of these patients was counted (see Figure 1 ). According to the time of progression to CRPC, HSPC was divided into 3 types:

[0057] (1) ADT-sensitive HSPC: most sensitive to ADT treatment, and the sensitive period is longer (≥60 months), and this part of patients accounts for about 20.5% (108 cases);

[0058] (2) ADT-resistant HSPC: the sensitive period to ADT is ≤12 months, that is, it progresses to CRPC, and this part of patients accounts for about 22.4% (118 cases);

[0059] (3) ADT-intermediate-sensitive HSPC: the sensitive period to ADT treatment is 12-60 months, and then it progresses to CRPC, and this part of patients accounts for about 57.1% (301 cases).

[0060] The paraffin-embedded tissue samples of 60 patients of the three types (20 patients in each group) were selected for whole protein 4D Label-free quantitative proteomics detection, and Figure 2The top 30 proteins with the largest expression differences were then verified using the targeted proteomics quantification technique PRM (parallel reaction monitoring). Through bioinformatics analysis, the 10 proteins with the highest sensitivity and specificity were selected through the Nomogram graph, and finally 10 protein molecules were screened for calculating each type of mHSPC. The 10 protein molecules include 7 protein molecules for evaluating the patient's drug resistance score: MCM4, NT5E, LTBP4, IAH1, HP, DOCK2, and EEF1A2, and 3 protein molecules for evaluating the patient's sensitivity score: TFF3, FABP5, and LAMP2.

[0061] The final scores of each protein molecule were analyzed using SPSS software to obtain a Normogram graph, and a formula was obtained by calculating the Normogram graph. According to the weights of the 10 protein molecules in the formula, the ROC curve and nomogram graph of the drug resistance score and the sensitivity score were drawn (see Figure 3 ). The AUC of the ROC curve of the drug resistance score was 0.92, and the AUC of the sensitivity score was 0.71, both with high sensitivity and specificity.

[0062] Example 2

[0063] A method for constructing a molecular typing model for predicting the sensitivity of hormone-sensitive prostate cancer patients to androgen deprivation therapy

[0064] (1) mHSPC patient diagnosis

[0065] Patients with metastatic prostate cancer who have not been treated with ADT are diagnosed based on imaging, PSA value, and clinical manifestations. The diagnosis is confirmed by pathological results of puncture biopsy. The diagnostic criteria must meet all the following conditions: 1) Imaging (MRI or whole body bone scan) suggests the presence of metastasis; 2) Prostate puncture biopsy pathology shows prostate cancer; 3) Not treated with ADT.

[0066] (2) Tissue sample acquisition

[0067] The patient's tissue sample was subjected to immunohistochemical staining. While the patient was undergoing puncture biopsy to obtain tissue for pathological diagnosis, a portion of the sample was also collected for paraffin block, sectioning, and preservation. After the pathological results confirmed prostate cancer, the collected tissue sections were used for the next step of immunohistochemistry.

[0068] (3) Immunohistochemistry

[0069] The tissue sections of mHSPC patients will be detected by immunohistochemistry for 10 molecular markers respectively. The FFPE tissue specimens are cut into 5 pm, deparaffinated in xylene, and passed through graded ethanol solutions. After heat-mediated, sodium citrate antigen retrieval (10 mM sodium citate, 0.05% Tween 20, pH = 6), the slides are incubated with the purchased antibodies of the target proteins and the HRP kit and DAB substrate kit. The sections are counterstained with hematoxylin and passed through graded dehydrating alcohol and xylene. The immunohistochemical sections are placed under a light microscope for observation, and the immunohistochemical intensity and positive cell density are used as the quantitative scoring criteria for interpretation. Then the expression of each protein molecule is scored according to the results of immunohistochemistry. The scoring criteria are as follows:

[0070] The staining intensity is graded by the IHC_Profiler function in the Image J software: the gray value of strong staining is [0-60], which is scored as 3 points; the gray value of moderate staining is (60-120], which is scored as 2 points; the gray value of weak staining is (120-180], which is scored as 1 point; the gray value of no staining is (180-236], which is scored as 0 point.

[0071] The positive rate is divided into 5 levels: no positive cells are scored as 0 points, <10% positive cells are scored as 1 point, 10%-50% positive cells are scored as 2 points, 51%-70% positive cells are scored as 3 points, and >70% positive cells are scored as 4 points. The positive rate is the percentage of positive cells in total cells.

[0072] Final score = Staining intensity x Positive rate Formula I

[0073] The results of immunohistochemistry are shown in Figure 3 wherein A is the 7 protein molecules of the drug resistance score of the three subtypes of mHSPC patients: MCM4, NT5E, LTBP4, IAH1, HP, DOCK2, and EEF1A2. B is the 3 protein molecules of the sensitivity score of the three subtypes of mHSPC patients: TFF3, FABP5, and LAMP2.

[0074] (4) ADT score calculation

[0075] The immunohistochemistry score of each protein molecule is substituted into the typing calculation model nomogram graph, and the drug resistance score (R Score) and the sensitivity score (S Score) are calculated according to Formula III and Formula IV, respectively, and the final ADT score is calculated according to Formula II.

[0076] ADT score = Drug resistance score - Sensitivity score Formula II.

[0077] Drug resistance score = -9.32942 + 1.74828 x MCM4 final score + 1.70248 x LTBP4 final score + 2.78289 x NT5E final score + 1.31930 x IAH1 final score + 3.65883 x DOCK2 final score - 0.11077 x HP final score - 1.71868 x EEF1A2 final score Formula III

[0078] Sensitivity score = -0.00090 - 0.22520 x TFF3 final score - 0.68322 x FABP5 final score + 0.55510 x LAMP2 final score Formula IV

[0079] (5) Molecular typing

[0080] According to the ADT score, the patients are typed as ADT sensitive (S group) when the score is <-1.5, as ADT resistant (R group) when the score is >0.5, and as ADT intermediate (I group) when the score is between -1.5 and 0.5.

[0081] Example 3

[0082] Verification experiment

[0083] In order to verify the accuracy of the prediction results of the molecular typing constructed in Example 2, 37 mHSPC patients in our hospital in the past 10 years were randomly selected for verification. According to the prediction score, 31 patients (83.8%) can accurately predict the time of progression to CRPC, that is, the sensitivity of the patients to ADT.

[0084] Table 1: Prediction results and actual results

[0085]

[0086]

[0087] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A protein composition for predicting the prognostic effect of metastatic hormone-sensitive prostate cancer, characterized in that, The protein composition consists of protein molecules used to assess patient drug resistance and protein molecules used to assess patient sensitivity; The protein molecules used to assess patient drug resistance consist of MCM4, NT5E, LTBP4, IAH1, HP, DOCK2, and EEF1A2. The protein molecules used to assess patient sensitivity consist of TFF3, FABP5, and LAMP2. The predicted prognosis of metastatic hormone-sensitive prostate cancer refers to the prediction of the sensitivity of patients with metastatic hormone-sensitive prostate cancer to androgen deprivation therapy or their survival time.

2. The use of a reagent for detecting the expression level of the protein composition of claim 1 in constructing a molecular subtyping model for predicting the sensitivity of metastatic hormone-sensitive prostate cancer patients to androgen deprivation therapy.

3. The application according to claim 2, characterized in that, The reagents include at least one of the following: reagents for protein immunoblotting, reagents for immunohistochemical detection, and reagents for in situ protein detection.

4. The application according to claim 2, characterized in that, When the reagent is an immunohistochemical detection reagent, the method for constructing a molecular subtyping model to predict the sensitivity of metastatic hormone-sensitive prostate cancer patients to androgen deprivation therapy includes the following steps: The staining intensity and positive rate of each protein molecule in the protein composition in the tissue samples of metastatic hormone-sensitive prostate cancer patients were detected, and the final score of each protein molecule was calculated according to Formula I. Final score = staining intensity × positive rate (Formula I) Substitute the final scores of each protein molecule into the nomogram of the typing calculation model to calculate the drug resistance score and the sensitivity score respectively, and calculate the ADT score according to Formula II. ADT score = Drug resistance score - Sensitivity score (Formula II) Based on the ADT score, the patient was molecularly classified: An ADT score < -1.5 indicates an ADT-sensitive type. An ADT score > 0.5 indicates ADT resistance. When the ADT score is between -1.5 and 0.5, it is considered an intermediate type of ADT. The drug resistance score is calculated according to Formula III; Drug resistance score = -9.32942 + 1.74828 × MCM4 final score + 1.70248 × LTBP4 final score + 2.78289 × NT5E final score + 1.31930 × IAH1 final score + 3.65883 × DOCK2 final score - 0.11077 × HP final score - 1.71868 × EEF1A2 final score Formula III; The sensitivity score is calculated according to formula IV; The sensitivity score is calculated as follows: -0.00090 -0.22520 × TFF3 final score - 0.68322 × FABP5 final score + 0.55510 × LAMP2 final score (Formula IV).

5. The application according to claim 4, characterized in that, The staining intensity is divided into four levels: no staining is 0 points, weak staining is 1 point, moderate staining is 2 points, and strong staining is 3 points. The staining intensity is distinguished by the grayscale value determined by the IHC_Profiler function in ImageJ software: the grayscale value of no staining is (180-236); the grayscale value of weak staining is (120-180); the grayscale value of moderate staining is (60-120); and the grayscale value of strong staining is [0-60].

6. The application according to claim 4, characterized in that, The positive rate is divided into 5 levels: 0 points for no positive cells, 1 point for <10% positive rate, 2 points for the positive rate in the range of [10%~50%], 3 points for the positive rate in the range of [50%~70%], and 4 points for the positive rate in the range of >70%.

7. The application according to claim 6, characterized in that, The positivity rate is the percentage of positive cells out of the total number of cells.

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