CN116246795A - Application of a model based on peripheral blood biomarkers in predicting the efficacy of upper gastrointestinal cancer chemotherapy - Google Patents

Abstract

The invention provides an application of a model constructed based on peripheral blood biomarkers in predicting upper gastrointestinal tumor chemotherapy efficacy. The peripheral blood biomarker is selected from hemoglobin, eosinophils and CD4 ⁺ T cell, memory CD4 ⁺ T cells, CD8 ⁺ T cells, CD4CD28 ⁺ T cells, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, CD8CD28 ⁺ One or more of T cells, ELR, leucocytes, platelets, neutrophils and monocytes, wherein the tumor of the upper digestive tract is esophageal cancer, gastric cancer or gastroesophageal junction cancer. The invention establishes a prediction model aiming at the upper gastrointestinal tumor chemotherapy curative effect, and the adopted peripheral blood detection index is low in cost, time-saving, convenient for clinical operation and dynamic monitoring, and has important clinical significance and wide applicability.

Description

Application of models based on peripheral blood biomarkers in predicting the efficacy of chemotherapy for upper gastrointestinal tumors

Technical Field

The present invention belongs to the field of biotechnology, and particularly relates to the application of a model constructed based on peripheral blood biomarkers in predicting the efficacy of chemotherapy for upper digestive tract tumors.

Background Art

Upper gastrointestinal tumors are the most common malignant tumors in the world, including esophageal cancer and gastric cancer. Surgical resection of early esophageal cancer and gastric cancer may be curative, but only 30-40% of patients have potentially resectable lesions when they seek medical treatment, that is, more than 60% of patients are already in the local advanced stage or have distant metastasis, and the prognosis is poor. For unresectable esophageal cancer and gastric cancer, the main treatment methods are currently chemotherapy, targeted therapy, immune checkpoint inhibitor therapy, etc.; for advanced esophageal cancer and gastric cancer, the efficacy of these treatments is limited. The median survival of metastatic esophageal cancer and gastric cancer after treatment is only about 10-13 months.

At present, chemotherapy is still the basic drug for all treatment options for patients with advanced or advanced esophageal cancer and gastric cancer. The main combination chemotherapy regimens are paclitaxel combined with platinum for esophageal cancer, and oxaliplatin and fluorouracil for gastric cancer. How to predict the efficacy of chemotherapy regimens has always been a difficult problem in clinical selection of treatment strategies. It is crucial to accurately evaluate the effectiveness of treatment and the risk of failure before receiving chemotherapy and to identify the population that benefits from chemotherapy for the treatment of upper gastrointestinal tumors. Currently, there are limited biomarkers for predicting the efficacy of chemotherapy. Some markers for predicting efficacy require peripheral blood free DNA or invasive tumor biopsy for detection, and expensive detection methods are used. It is difficult to implement dynamic monitoring and clinical application is limited. Therefore, it is of great research significance to select reliable and simple biomarkers or efficacy prediction models.

Summary of the invention

In order to make up for the shortcomings of the existing technology and find reliable and simple biomarkers that can predict the efficacy of chemotherapy for upper gastrointestinal tumors (such as esophageal cancer, gastric cancer, and gastroesophageal junction cancer), the present invention provides an efficacy prediction model based on peripheral blood cells, biochemical indicators, lymphocyte subpopulations and other indicators, which can well predict the efficacy of chemotherapy for upper gastrointestinal tumors and provide an economical and time-saving method for prediction.

The first aspect of the present invention provides an application of a reagent for detecting peripheral blood biomarkers in a sample in the preparation of a product for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors, wherein the peripheral blood biomarkers are selected from one or more of hemoglobin, eosinophils, CD4 ⁺ T cells, memory CD4 ⁺ T cells, CD8 ⁺ T cells, CD4CD28 ⁺ T cells, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, CD8CD28 ⁺ T cells, ELR, leukocytes, platelets, neutrophils, and monocytes.

Furthermore, the upper digestive tract tumor is esophageal cancer, gastric cancer or gastroesophageal junction cancer.

Preferably, the upper digestive tract tumor is esophageal squamous cell carcinoma, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

Furthermore, the evaluation levels of chemotherapy efficacy are divided into partial remission, disease stabilization and disease progression (refer to Response Evaluation Criteria in Solid Tumors 1.1 (RECISTv1.1)). Among them, partial remission and/or disease stabilization are evaluated as achieving disease control.

In one embodiment of the present invention, the chemotherapy regimen for esophageal cancer is a treatment mainly based on paclitaxel combined with platinum drugs.

In one embodiment of the present invention, the chemotherapy regimen for gastric cancer is a treatment with oxaliplatin combined with fluorouracil drugs (such as S-1 or capecitabine).

In one embodiment of the present invention, hemoglobin, eosinophils, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, memory CD4 ⁺ T cells, and CD4CD28 ⁺ T cells in the peripheral blood biomarkers can be used as a combined indicator to predict and/or evaluate the efficacy of chemotherapy for esophageal cancer. The higher the content of the peripheral blood biomarkers, the easier it is for the efficacy to achieve partial remission and/or disease stabilization (disease control).

In another embodiment of the present invention, the eosinophils, CD8 ⁺ T cells, memory CD4 ⁺ T cells, and ELR in the peripheral blood biomarkers can be used as a combined indicator to predict and/or evaluate the chemotherapy efficacy of esophageal cancer. The higher the content of the peripheral blood biomarkers, the easier it is for the efficacy to achieve partial remission or even complete remission.

In one embodiment of the present invention, the eosinophils, memory CD4 ⁺ T cells, and ELR in the peripheral blood biomarkers can be used as a combined indicator to predict and/or evaluate the efficacy of chemotherapy for gastric cancer. The higher the content of the peripheral blood biomarkers, the easier it is for the efficacy to achieve partial remission and/or disease stabilization (disease control).

In another embodiment of the present invention, the leukocytes, platelets, neutrophils, monocytes, and CD8CD28 ⁺ T cells in the peripheral blood biomarkers can be used as a combined indicator to predict and/or evaluate the efficacy of chemotherapy for gastric cancer. The higher the content of the peripheral blood biomarkers, the easier it is for the efficacy to achieve partial remission or even complete remission.

Furthermore, the sample is peripheral blood obtained from a biopsy subject.

Furthermore, the subject is a mammal, particularly a human.

Furthermore, the product for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors includes a product for detecting the content of the peripheral blood biomarker.

Furthermore, the product for detecting the content of the peripheral blood biomarker may be a reagent, a test kit, a test paper or an instrument platform.

Furthermore, the product for detecting the content of the peripheral blood biomarker can be based on the principle of cell counting technology, for example, resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductivity light scattering combined detection method, impedance radio frequency technology combined detection method, light scattering and cell chemistry technology, etc.

The second aspect of the present invention provides a product for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors, and the product for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors includes a product for detecting the content of the peripheral blood biomarkers described in the first aspect.

Furthermore, the upper digestive tract tumor is esophageal cancer, gastric cancer or gastroesophageal junction cancer.

Preferably, the upper digestive tract tumor is esophageal squamous cell carcinoma, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

Furthermore, the evaluation levels of chemotherapy efficacy are divided into partial remission, disease stabilization and disease progression, wherein partial remission and/or disease stabilization are evaluated as reaching the disease control level.

Furthermore, the product for detecting the content of the peripheral blood biomarker may be a reagent, a test kit, a test paper or an instrument platform.

Furthermore, the product for detecting the content of the peripheral blood biomarker can be based on the principle of cell counting technology, for example, resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductivity light scattering combined detection method, impedance radio frequency technology combined detection method, light scattering and cell chemistry technology, etc.

The third aspect of the present invention provides a prediction model for the efficacy of chemotherapy for upper gastrointestinal tumors, wherein the prediction model uses the peripheral blood biomarkers described in the first aspect as variables.

Furthermore, the upper digestive tract tumor is esophageal cancer, gastric cancer or gastroesophageal junction cancer.

Preferably, the upper digestive tract tumor is esophageal squamous cell carcinoma, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

Furthermore, the evaluation levels of chemotherapy efficacy are divided into partial remission, disease stabilization and disease progression, wherein partial remission and/or disease stabilization are evaluated as achieving disease control.

In one embodiment of the present invention, the variables of the prediction model for the efficacy of chemotherapy for esophageal cancer are hemoglobin, eosinophils, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, memory CD4 ⁺ T cells and a combination of CD4CD28 ⁺ T cells.

In another embodiment of the present invention, the variables of the prediction model for the efficacy of chemotherapy for esophageal cancer are a combination of eosinophils, CD8 ⁺ T cells, memory CD4 ⁺ T cells and ELR.

In one embodiment of the present invention, the variables of the prediction model for the efficacy of chemotherapy for gastric cancer are a combination of eosinophils, memory CD4 ⁺ T cells and ELR.

In another embodiment of the present invention, the variables of the prediction model for the efficacy of chemotherapy for gastric cancer are a combination of leukocytes, platelets, neutrophils, monocytes and CD8CD28 ⁺ T cells.

A fourth aspect of the present invention provides a method for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors, which comprises the step of detecting a reagent for the peripheral blood biomarker described in the first aspect.

Furthermore, the upper digestive tract tumor is esophageal cancer, gastric cancer or gastroesophageal junction cancer.

Preferably, the upper digestive tract tumor is esophageal squamous cell carcinoma, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

Furthermore, the evaluation levels of chemotherapy efficacy are divided into partial remission, disease stabilization and disease progression, wherein partial remission and/or disease stabilization are evaluated as reaching the disease control level.

Furthermore, the method comprises the following steps:

(1) Obtaining samples from subjects;

(2) detecting the content of the peripheral blood biomarkers in the subject's sample;

(3) Correlating the measured content of the peripheral blood biomarker with the chemotherapy efficacy of the subject's upper gastrointestinal tumor (such as esophageal cancer, gastric cancer, gastroesophageal junction adenocarcinoma).

Furthermore, the sample is peripheral blood obtained from a biopsy subject.

Furthermore, the subject is a mammal, particularly a human.

Furthermore, the criteria for predicting and/or evaluating the efficacy of chemotherapy for upper gastrointestinal tumors are: detecting the content of the peripheral blood biomarkers, and compared with the reference, the higher the content of the peripheral blood biomarkers of the subject before chemotherapy, the easier it is for the chemotherapy efficacy of the upper gastrointestinal tumor to reach the level of disease control (such as partial remission and/or disease stabilization), and vice versa.

Furthermore, the reference is the content value corresponding to the maximum Youden index of the peripheral blood biomarker of the subject before chemotherapy.

In one embodiment of the present invention, the higher the hemoglobin, eosinophils, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, memory CD4 ⁺ T cells, and CD4CD28 ⁺ T cell contents, the easier it is for the chemotherapy effect of esophageal cancer to achieve partial remission and/or disease stabilization (disease control).

In another embodiment of the present invention, the higher the content of eosinophils, CD8 ⁺ T cells, memory CD4 ⁺ T cells, and ELR, the easier it is for the chemotherapy effect of esophageal cancer to achieve partial remission.

In one embodiment of the present invention, the higher the content of eosinophils, memory CD4 ⁺ T cells, and ELR, the easier it is for the chemotherapy effect of gastric cancer to achieve partial remission and/or disease stabilization (disease control).

In another embodiment of the present invention, the higher the content of leukocytes, platelets, neutrophils, monocytes, and CD8CD28 ⁺ T cells, the easier it is for the chemotherapy effect of gastric cancer to achieve partial remission.

The present invention provides a peripheral blood biomarker for predicting the efficacy of chemotherapy for upper gastrointestinal tumors (such as esophageal cancer, gastric cancer, and gastroesophageal junction cancer), which can be used for auxiliary diagnosis to predict the risk of chemotherapy for upper gastrointestinal tumors, with good detection specificity and high sensitivity. The peripheral blood index detection used in the present invention is a conventional kit, which is low-cost, time-saving, convenient for clinical operation and dynamic monitoring, and has important clinical significance and wide applicability. The peripheral blood combined index of the present invention reflects that some inflammatory factors, peripheral blood cells and lymphocyte subpopulations are closely related to the development of tumors, and interact with the body's immune microenvironment, and is expected to be used as an evaluation index for monitoring, predicting and judging the prognosis of upper gastrointestinal tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the ROC curve of the combined indicator model A for predicting the efficacy of chemotherapy for esophageal cancer to achieve disease control.

Figure 2 shows the ROC curve of the combined indicator model B for predicting the efficacy of chemotherapy for esophageal cancer to achieve partial disease remission.

FIG3 shows the ROC curve of the combined indicator model C for predicting the efficacy of chemotherapy for gastric cancer to achieve disease control.

FIG4 shows the ROC curve of the combined indicator model D for predicting the efficacy of chemotherapy for gastric cancer to achieve partial disease remission.

DETAILED DESCRIPTION

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

The term "Youden Index" is a method for evaluating the authenticity of a screening test, which indicates the total ability of the screening method to detect true patients and non-patients. The Youden Index is the sum of sensitivity and specificity minus 1. The cutoff point corresponding to the maximum Youden Index is used as the CUT OFF value to ensure the optimal accuracy of the test kit.

The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

Example 1

1.1 Research subjects

A total of 92 patients with upper gastrointestinal tumors (38 cases of esophageal cancer and 54 cases of gastric cancer) were prospectively enrolled in this study.

Inclusion criteria: (1) aged over 18 years; (2) esophageal squamous cell carcinoma, gastric or gastroesophageal junction adenocarcinoma confirmed by pathological histology or cytology; (3) locally advanced stage or stage IV (AJCC 7.0) at the time of enrollment; (4) no surgery or other anti-tumor treatment within 6 months before enrollment; (5) normal organ function; (6) meeting the requirements for chemotherapy; (7) voluntary participation in this study.

1.2 Research Methods

1.2.1 Treatment plan and efficacy evaluation

Clinical researchers selected standard chemotherapy regimens: paclitaxel combined with platinum was mainly used for esophageal squamous cell carcinoma; oxaliplatin combined with fluorouracil drugs (Tegafur or capecitabine) was mainly used for gastric and gastroesophageal junction adenocarcinoma.

Efficacy evaluation: With reference to Response Evaluation Criteria in Solid Tumors 1.1 (RECIST v1.1), efficacy evaluation was performed every 6 weeks; the efficacy evaluation grades were divided into partial response (PR), stable disease (SD), and progressive disease (PD); both partial response and stable disease were evaluated as achieving disease control.

1.2.2 Test items

Peripheral blood was collected from patients within 1 week before anti-tumor treatment (baseline) for examination of complete blood cell analysis, lymphocyte subsets, erythrocyte sedimentation rate (ESR), hypersensitive C-reactive protein (hsCRP), albumin ALB/prealbumin preALB, lymphocyte subsets, etc. The combined marker levels (MLR, NLR, ELR, BLR, PLR, CAR, COP, CLR, CBR) were calculated for each patient.

MLR=monocyte count/lymphocyte count; NLR=neutrophil count/lymphocyte count; ELR=eosinophil count/lymphocyte count; BLR=basophil count/lymphocyte count; PLR=platelet count/lymphocyte count; CAR=CRP/ALB; COP=CRP/preALB; CLR=CRP/lymphocyte; CBR=CRP/BMI.

1.3 Statistical analysis

SPSS 26.0 was used for statistical analysis. T test and ANOVA analysis of variance were used to compare the differences in data between groups. Mann-Whitney and Kruskal-Wallis tests were used for nonparametric tests. Kaplan-Meier survival analysis was used to compare the differences in survival, and ROC analysis was used to determine the efficacy of marker differences as diagnostic criteria. Two-way P < 0.05 was set to indicate that the difference was statistically significant. GraphPad Prism 8.0 software (San Diego, USA) was used for statistical analysis and drawing, and Mann-Whitney and Kruskal-Wallis rank sum tests were used to compare the differences in clinical data.

1.4 Clinical characteristics of enrolled patients

Table 1 Clinical characteristics of patients with esophageal cancer and gastric cancer

Table 2 Circulatory parameters of patients with esophageal cancer and gastric cancer

1.5 Single indicator screening

Table 3 Screening results of esophageal cancer indicators

Table 4 Gastric cancer index screening results

As shown in Table 3, the single indicator screening for disease control achieved by early esophageal cancer chemotherapy found that hemoglobin (Hb), eosinophils (EOS), CD4CD28 ⁺ T/CD4 ⁺ T cells, memory CD4 ⁺ T, and CD4CD28 ⁺ T cells had significant differences; the single indicator screening for partial remission achieved by early esophageal cancer chemotherapy found that eosinophil (EOS) levels, CD8 ⁺ T cell levels, memory CD4 ⁺ T, and ELR had significant differences (P<0.05).

As shown in Table 4, the single indicator screening for disease control achieved by early gastric cancer chemotherapy found that eosinophils (EOS), memory CD4 ⁺ T, and ELR had significant differences; the single indicator screening for partial remission achieved by early gastric cancer chemotherapy found that white blood cell (WBC) levels, platelet (PLT) levels, neutrophils (NEUT), monocytes (Mono), and CD8CD28 ⁺ T cells had significant differences (P < 0.05).

Example 2 Screening of peripheral blood markers related to chemotherapy efficacy of esophageal cancer and construction of a prediction model

Through early single-indicator screening, it was found that the higher the hemoglobin (Hb), eosinophils (EOS), CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, memory CD4 ⁺ T, and CD4CD28 ⁺ T cell counts, the easier it is to achieve disease control (partial remission + disease stabilization) with esophageal cancer chemotherapy, and the less likely it is to progress.

The above single indicators with efficacy prediction value were combined to construct an efficacy prediction model, and their predictive effects were evaluated by ROC diagnostic analysis. The results showed that their areas under the receiver operating characteristic curve (AUROC) were between 0.4210 and 0.7920.

The area under the curve of the combined indicator model A for predicting the efficacy of chemotherapy for esophageal cancer to achieve disease control (partial remission PR + stable disease SD) was 79.2% (Figure 1), with a sensitivity of 82.6%, a specificity of 80%, p=0.012 (95CI 0.609-0.975), which was better than the single indicator prediction efficiency.

It was also found that the higher the eosinophil (EOS) level, CD8 ⁺ T cell level, memory CD4 ⁺ T count, and ELR, the easier it was to achieve partial remission (PR). The single indicators with efficacy (PR) prediction value were combined to construct an efficacy prediction model, and their predictive effects were evaluated by ROC diagnostic analysis. The results showed that their area under the receiver operating characteristic curve (AUROC) was between 0.701 and 0.851.

The area under the curve of the combined indicator model B for predicting the efficacy of chemotherapy for esophageal cancer to achieve partial remission (PR) was 85.1% (Figure 2), with a sensitivity of 77.8%, a specificity of 89.7%, p=0.002 (95CI 0.691-1.000), which was better than that of a single indicator.

Example 3 Screening of peripheral blood markers related to chemotherapy efficacy in gastric cancer and construction of a prediction model

Through early single-indicator screening, it was found that the higher the eosinophils (EOS), memory CD4 ⁺ T, and ELR, the easier it is to achieve disease control (partial remission + disease stabilization) with chemotherapy for gastric cancer, and the less likely it is to progress.

The above single indicators with efficacy prediction value were combined to construct an efficacy prediction model, and their prediction effects were evaluated by ROC diagnostic analysis. The results showed that their areas under the receiver operating characteristic curve (AUROC) were between 0.722 and 0.936.

The area under the curve of the combined indicator model C for predicting the efficacy of chemotherapy for gastric cancer to achieve disease control (partial remission PR + stable disease SD) was 93.6% (Figure 3), with a sensitivity of 84.1% and a specificity of 90%, p<0.001 (95CI 0.861-1.000), which was better than the single indicator prediction efficiency.

The higher the white blood cell (WBC) level, platelet (PLT) level, neutrophil (NEUT) count, monocyte (Mono), and CD8CD28 ⁺ T cells, the easier it is to achieve partial remission (PR).

The single indicators with predictive value for efficacy (PR) were combined to construct a efficacy prediction model, and their predictive effects were evaluated by ROC diagnostic analysis. The results showed that the AUROC ranged from 0.671 to 0.807.

The area under the curve of the combined indicator model D for predicting the efficacy of chemotherapy for gastric cancer to achieve partial remission (PR) was 80.7% (Figure 4), with a sensitivity of 88.9%, a specificity of 66.7%, p<0.001 (95CI 0.692-0.922), and better predictive efficiency than a single indicator.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The application of a reagent for detecting peripheral blood biomarkers in a sample in the preparation of a product for predicting and/or evaluating the curative effect of upper gastrointestinal tumor chemotherapy, characterized in that, the peripheral blood biomarkers are selected from hemoglobin, hemotropin Acid cells, CD4 ⁺ T cells, memory CD4 ⁺ T cells, CD8 ⁺ T cells, CD4CD28 ⁺ T cells, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, CD8CD28 ⁺ T cells, ELR, white blood cells, platelets, neutrophils, One or more of monocytes. 2. The application according to claim 1, characterized in that the upper gastrointestinal tumor is esophageal cancer, gastric cancer or gastroesophageal junction cancer. 3. The application according to claim 2, wherein the upper gastrointestinal tumor is esophageal squamous cell carcinoma, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. 4. The application according to claim 1, wherein the sample is peripheral blood obtained from a biopsy subject. 5 . The application according to claim 1 , wherein the product for predicting and/or evaluating the curative effect of upper gastrointestinal tumor chemotherapy includes a product for detecting the content of the peripheral blood biomarker. 6. The application according to claim 5, wherein the product for detecting the content of the peripheral blood biomarker is a reagent, a kit, a test strip or an instrument platform. 7. The application according to any one of claims 1-6, wherein the peripheral blood biomarkers are hemoglobin, eosinophils, CD4CD28 ⁺ T/CD4 ⁺ T cell ratio, memory CD4 ⁺ T cell and CD4CD28 ⁺ T cells, the upper gastrointestinal tumor is esophageal cancer. 8. The application according to any one of claims 1-6, characterized in that, the peripheral blood biomarkers are eosinophils, CD8 ⁺ T cells, memory CD4 ⁺ T cells and ELR, and the upper digestion Tract tumor is esophageal cancer. 9. The application according to any one of claims 1-6, wherein the peripheral blood biomarkers are eosinophils, memory CD4 ⁺ T cells and ELR, and the upper gastrointestinal tumor is gastric cancer. 10. The application according to any one of claims 1-6, characterized in that, the peripheral blood biomarkers are white blood cells, platelets, neutrophils, monocytes and CD8CD28 ⁺ T cells, and the above Gastrointestinal tumors are stomach cancers.

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