Exploration Study of Molecular Biomarkers for Tumor-related Anxiety and Depression

Exploration Research on Molecular Markers of Tumor-Related Anxiety and Depression I.Research Background Tumors are major diseases threatening the health of the Chinese population. In 2020, China reported 4.57 million new cases and 3 million deaths from malignant tumors, both ranking first globally [1]. Tumor-related anxiety and depression are psychological and mental disorders affecting 30%-70% of tumor patients, ranking as one of the most common comorbidities in tumor clinical settings. Annually, over 10 million tumor patients globally experience reduced treatment adherence and lower quality of life due to tumor-related psychological disorders [2]. Tumor-related anxiety and depression pose significant harm to patients, not only exacerbating their physical discomfort but also significantly reducing treatment adherence and clinical efficacy, increasing the risk of tumor recurrence and metastasis, and directly leading to poorer long-term survival outcomes. However, to date, there is still a lack of specific early screening molecular markers for tumor-related anxiety and depression in clinical practice. Current intervention strategies primarily rely on symptomatic psychological counseling and non-specific anti-anxiety and antidepressant medications, lacking targeted and precise intervention approaches, resulting in many patients' psychological disorders not being identified or effectively treated early. The field of tumor psychological diagnosis and treatment in China is underdeveloped, with an incomplete clinical diagnostic system, showing a significant gap compared to developed Western countries. Surgery, radiotherapy, chemotherapy, and immunotherapy are the main clinical treatment methods for malignant tumors. Due to the complex pathogenesis of tumors, diverse pathological types, and individual differences in patients' physiological conditions and psychological tolerance, the onset, severity, and intervention outcomes of tumor-related anxiety and depression exhibit significant heterogeneity. There remains substantial room for improvement in the precise screening, risk stratification, and targeted intervention of these psychological disorders. Building on the extensive clinical tumor samples from the Chinese population and molecular mechanism research data accumulated by our team [3], as well as the foundational research confirming that tumor-derived proteins can mediate central nervous system inflammation and emotional abnormalities, there are currently no reported clinical studies on the correlation between tumor-derived proteins and the levels of anxiety and depression in tumor patients. It is urgent to conduct clinical research to clarify the intrinsic relationship between the two, providing new molecular targets and clinical evidence for the early screening and precise intervention of tumor-related anxiety and depression.

Main References

• Hyuna Sung., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries, CA CANCER J CLIN 2021;71:209-249.
• Vita G., et al., Antidepressants for the treatment of depression in people with cancer. Cochrane Database Syst Rev. 2023 Mar 31;3(3):CD011006.
• Xu Chen., et al. GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia. Signal Transduction and Targeted Therapy. 2024, 26;9(1):253.

II.Research Objectives and Significance

• Research Objectives To identify and validate molecular biomarkers associated with tumor-related anxiety and depression. By integrating psychological assessment data from clinical cancer patients with molecular detection results, and utilizing clinically accessible samples such as tumor tissues and sera from clinical cohorts, this study aims to clinically validate the potential regulatory tumor-derived proteins previously identified by our team. The goal is to clarify the clinical value of tumor-derived proteins as molecular biomarkers for tumor-related anxiety and depression, providing a molecular basis for early screening and risk stratification of these conditions. Alternatively, it seeks to establish a tumor-related anxiety and depression risk assessment model based on the expression levels of tumor-derived proteins, offering a reference for the precise identification and targeted intervention of psychological disorders in cancer patients.
• Research Significance The research outcomes will yield proprietary intellectual property rights for novel molecular targets and associated biomarkers for the screening and intervention of tumor-related anxiety and depression, and promote their translation into clinical diagnostic and therapeutic applications. This study will provide critical data for classifying cancer patients into psychological intervention tiers based on their anxiety and depression risk levels, supporting the precise screening and personalized intervention of psychological disorders in clinical oncology, and contributing to the enhancement of the comprehensive cancer treatment system.

III.Implementation Plan

• Inclusion/Exclusion Criteria 1.1. Inclusion Criteria

• Patients with solid tumors aged ≥18 years and an expected survival period of ≥3 months;
• Patients who meet clinical diagnostic criteria and are pathologically/histologically confirmed as having solid tumors, including liver cancer patients (diagnosable by imaging);
• Patients who can complete standardized stratified assessments for tumor-related anxiety and depression, and are able to cooperate with researchers in completing psychological scales such as the Self-Rating Anxiety Scale (SAS) and the Self-Rating Depression Scale (SDS). They have not received any anti-anxiety/depression medications, professional psychological counseling, or psychiatric interventions before scale assessment;
• Patients with clear consciousness, normal language communication, comprehension, and cognitive abilities, without a history of psychiatric disorders, and who can independently provide feedback on research-related information and cooperate with follow-up;
• Patients who voluntarily participate in the study, fully understand the research objectives, procedures, potential risks, and benefits, and have signed a written informed consent form;
• Patients who can cooperate in completing the required laboratory tests (complete blood count, blood biochemistry, coagulation function, etc.) and clinical data collection before and during the perioperative period to ensure the completeness of research data.

1.2. Exclusion Criteria

• Pregnant or lactating women;
• Patients aged <21 years at first visit and with an expected survival period of <3 months;
• Patients who have previously received anti-anxiety/depression medications or professional psychological interventions;
• Patients with a history or current diagnosis of psychiatric disorders, including schizophrenia, bipolar disorder, severe cognitive dysfunction, mental retardation, or those who cannot cooperate with SAS/SDS scale assessment or have contraindications to scale evaluation;
• Patients with severe organ dysfunction or severe underlying diseases, including: liver failure (Child-Pugh C grade), renal failure (serum creatinine >250 μmol/L or >2.83 mg/dL), New York Heart Association (NYHA) Class IV heart failure, active pulmonary tuberculosis, HIV infection, etc. (to be determined);
• Patients with contraindications to tumor tissue or blood sample collection, including coagulation disorders (INR >1.5, platelets <50×10⁹/L), severe bleeding tendency, or surgical specimens that cannot meet detection requirements (e.g., >50% necrotic tissue, insufficient tissue amount);
• Patients with emotional abnormalities due to non-tumor factors, including hyperthyroidism/hypothyroidism, severe malnutrition, chronic wasting diseases, or psychological stress disorders;
• Patients who have undergone non-tumor-related surgical procedures, enteral/parenteral nutrition support within the past 1 month, or have clinical symptoms such as gastrointestinal mechanical obstruction, intractable vomiting, ascites, significant edema, or large pleural effusion;
• Patients who are planned or currently using medications that may affect emotional assessment or protein expression detection during the study, including: long-term oral corticosteroids, 5-HT receptor agonists/antagonists (SSRIs, etc., even short-term use within 2 weeks before sampling is excluded), antipsychotic drugs, or gestagenic synthetic steroid derivatives (short-term inhaled/local use of steroids or intermittent use of inhaled bronchodilators are excluded);
• Patients with incomplete clinical data, inability to cooperate with research-related follow-up and testing, or those who refuse to sign the informed consent form or have poor compliance.

• Recruitment Procedure This study does not involve outpatients recruitment. The blood and tumor tissues selected from clinical patients are all derived from hospitalized patients. The blood is the remaining portion after routine blood tests, and the tumor tissues are the remaining portions collected after surgery, with priority given to ensuring no impact on pathology diagnosis.The remaining tissue samples and blood analyses will be stored in the -80°C freezer in this department for subsequent new scientific research.

• Research Process In the field of comprehensive tumor diagnosis and treatment, achieving precise screening and targeted intervention for psychological disorders is a key goal to improve the quality of life of tumor patients and enhance the comprehensive management system for tumor treatment. Malignant tumors, as systemic diseases, not only affect patients physically but also easily trigger psychological and mental disorders such as anxiety and depression. The high incidence and concealment of tumor-related anxiety and depression make them an important yet often overlooked aspect in tumor clinical diagnosis and treatment. This project fully leverages the rich clinical resources and molecular detection platforms of the collaborating institutions to conduct in-depth analysis and stratification of solid tumor patients from the perspective of molecular expression - psychological phenotype association. It aims to explore molecular markers that can precisely correlate with the levels of anxiety and depression in tumor patients, as well as detection indicators suitable for clinical screening, thereby promoting the precise identification and intervention of tumor-related anxiety and depression and improving the comprehensive tumor diagnosis and treatment system.

• Research Subjects and Sample Size Determination The subjects of this study are solid tumor patients, with no additional healthy control group. A total of 500 solid tumor patients meeting the inclusion criteria will be enrolled, and they will be divided into an anxiety and depression group and a non-anxiety and depression group based on the scores from the anxiety and depression scale. In determining the sample size, the research team used a scientifically rigorous calculation method, employing the MedCalc software to calculate the sample size based on the AUC value. The specific parameters are set as follows: estimated AUC = 0.9, significance level α = 0.05, and power 1-β = 0.9. The null hypothesis is set as AUC = 0.8. Considering clinical realities, the incidence of anxiety and depression in tumor patients is approximately 30%-50%. Through precise calculations, it is determined that at least 50 patients in the anxiety and depression group and at least 50 patients in the non-anxiety and depression group should be enrolled, with a total sample size of 100. This is sufficient to meet the statistical requirements for the correlation analysis of molecular markers and anxiety and depression levels in this study, ensuring the reliability and representativeness of the results.
• Research Foundation and Preliminary Exploration Before the study began, the research team had completed basic research on tumor-derived proteins, confirming that tumor-derived proteins can regulate anxiety and depression by mediating central nervous system inflammation. Additionally, serum and tumor tissue samples from some solid tumor patients were retrieved from the clinical sample bank. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence techniques were used to quantitatively measure the levels of tumor-derived proteins in serum and to locate and detect the expression levels of tumor-derived proteins in tissues, respectively. The ELISA technique, based on the specific binding of antigens and antibodies, achieves precise quantification of protein levels through steps such as coating with primary antibody, sample incubation, binding with enzyme-labeled secondary antibody, and substrate color development, utilizing standard curves for accurate quantification. Immunofluorescence technology, on the other hand, uses specific fluorescent antibodies for labeling and observes the localization and expression intensity of proteins in tumor tissues using laser confocal microscopy for semi-quantitative analysis. The preliminary experimental results of these studies have laid a solid technical and data foundation for this research, established standardized experimental procedures for tumor-derived protein detection, and provided important references for subsequent large-scale clinical validation.
• Collection of Clinical Data and Multifactorial Correlation Analysis Collecting comprehensive and accurate clinical data and psychological assessment data is a crucial aspect of this study.The research team will systematically collect and standardize the clinical data of enrolled patients, covering three dimensions: first, basic patient information including age, gender, height, weight, BMI, education level, marital status, smoking and drinking history, medical history, and drug allergy history; second, tumor-related clinical information including tumor type, pathological histology type, tumor differentiation degree, TNM stage, tumor size, lymph node metastasis status, etc.; third, research-specific assessment information including the HADS score results, scale assessment time, and sample collection time. Additionally, laboratory test results such as complete blood count, blood biochemistry, coagulation function, and tumor markers will be collected at the time of enrollment to ensure the completeness of clinical data. Through multivariate correlation analysis of the above data and statistical methods such as Pearson/Spearman correlation analysis and logistic regression analysis, confounding factors such as age, tumor type, and tumor stage will be adjusted to identify factors closely related to the anxiety and depression levels of tumor patients, and to clarify the association and independent effect of tumor-derived protein expression levels with tumor-related anxiety and depression.
• Validation of cohort study design and implementation The validation cohort in this study plays a crucial role in verifying the association between tumor-derived proteins and the anxiety and depression levels of tumor patients, as well as evaluating the clinical efficacy of tumor-derived proteins as molecular markers for tumor-related anxiety and depression. The study prospectively enrolls 100 newly diagnosed solid tumor patients undergoing surgical treatment between 2026-2027. The selection of study subjects strictly follows the predefined inclusion and exclusion criteria. Inclusion criteria include: pathological/histological/cytological confirmation of solid tumors; eligibility for surgical resection; ability to complete the Hospital Anxiety and Depression Scale (HADS) assessment and required sample collection and related tests; and voluntary participation in the study with signed informed consent. Exclusion criteria cover: concurrent severe dysfunction of vital organs such as the heart, liver, and kidneys; distant extensive tumor metastasis; history of other malignant tumors; presence of psychiatric diseases or cognitive communication disorders, unable to complete the HADS assessment; contraindications to sample collection, unable to provide qualified tumor tissue and serum samples, etc.

Regarding sample collection, tumor tissue samples and peripheral serum samples are collected simultaneously 1-3 days before surgery (before systemic tumor treatment). The tumor tissue sample is obtained during surgical resection, ensuring a tumor parenchyma volume of ≥0.5 cm³. Approximately 5 mL of fasting peripheral venous blood is collected for serum samples without anticoagulants. After collection, the serum samples are allowed to stand at room temperature for 30-60 minutes to ensure complete blood coagulation, then centrifuged at 3000 rpm for 15 minutes to separate the serum. The tumor tissue samples are rinsed with sterile PBS buffer and then treated with 4% paraformaldehyde solution and liquid nitrogen. The prepared tissue and serum samples are stored at -80°C for subsequent testing. Immunofluorescence techniques are used to quantitatively detect the expression levels of tumor-derived proteins in tumor tissues, while ELISA is employed to quantitatively measure the levels of tumor-derived proteins in serum. By testing tumor-derived proteins in samples obtained during the perioperative baseline period and combining the concurrent HADS assessment results, the intrinsic association between tumor-derived protein expression levels and the anxiety and depression phenotypes of tumor patients can be precisely analyzed, thereby accurately evaluating its clinical application value as a molecular marker for tumor-related anxiety and depression.

• Observation Indicators and Follow-up Plan A. Initial and Subsequent Follow-up Testing Indicators During the initial consultation and subsequent follow-up of patients, a series of comprehensive tests and evaluations are required. Psychological status assessment is the core observation indicator. The Hospital Anxiety and Depression Scale (HADS) score is the key basis for assessing the level of anxiety and depression in patients. The changes in the anxiety dimension (HADS-A) and depression dimension (HADS-D) scores can intuitively reflect the dynamic changes in the patient's emotional state, while the classification results of anxiety and depression provide standardized references for judging the severity of psychological disorders. By regularly completing HADS scale assessments, it is possible to promptly identify fluctuations in patients' psychological states, providing precise evidence for clinical intervention in tumor-related anxiety and depression.

Laboratory retention tests include complete blood count, blood biochemistry, tumor markers, and other indicators. Complete blood count can reflect changes in the quantity and morphology of blood cells such as red blood cells, white blood cells, and platelets, helping to understand the patient's hematopoietic function, the presence of infection or postoperative inflammatory reactions, etc. Blood biochemistry indicators cover liver function, renal function, electrolytes, albumin, etc., reflecting the overall metabolic status and organ function level of the patient, and assessing the patient's postoperative physical recovery. Tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are of great significance for monitoring the risk of tumor recurrence and metastasis after surgery and evaluating the progression of the tumor condition.

Tumor imaging is an important means of assessing the patient's tumor condition. Based on the patient's tumor type and clinical diagnostic needs, examination methods such as chest CT, abdominal CT, and ultrasound are selected. Through imaging, doctors can clearly observe the postoperative tumor resection status, the recovery of the surgical area, and the presence of local recurrence or distant metastasis. During the postoperative follow-up, imaging examinations can intuitively assess the stability of the tumor condition, providing important imaging evidence for adjusting subsequent tumor clinical treatment plans.

B.Patient-Reported Outcome

Indicators Patient-reported outcome indicators involve psychological assessment, laboratory tests, and tumor condition assessment, providing a comprehensive and integrated evaluation of the patient's psychological state, physical function, and tumor condition, and offering multidimensional references for the clinical efficacy validation of tumor-derived protein molecular markers.

Psychological assessment indicators are centered on the Hospital Anxiety and Depression Scale (HADS), including the total HADS score, anxiety dimension score, depression dimension score, and the classification results of anxiety and depression, which can accurately reflect the occurrence, development, and improvement of tumor-related anxiety and depression after intervention, serving as direct evidence for assessing the changes in psychological status. Laboratory test indicators include changes in the values of complete blood count, blood biochemistry, and tumor markers, not only reflecting the recovery of the patient's physical function after surgery but also excluding interference from physical diseases or tumor progression in the patient's psychological state, providing data support for analyzing the pure correlation between tumor-derived protein and anxiety and depression. Tumor condition assessment indicators include postoperative pathology results and imaging examination results. Postoperative pathology can clarify the basic information of the tumor, such as its pathological type, differentiation degree, and lymph node metastasis. Imaging examinations dynamically reflect the risk of tumor recurrence and metastasis after surgery. The combination of these two can define the impact of tumor clinical characteristics on the patient's psychological state, providing a basis for subgroup analysis of the expression of tumor-derived protein and its correlation with anxiety and depression in patients with different tumor characteristics.

C.Follow-up System Tracking Records

To monitor the patient's psychological state, physical recovery, and tumor condition changes in a long-term and systematic manner, the study uses an information-based follow-up system to track and record patients for 6 months post-surgery. The follow-up frequency is once at 1 month, 3 months, and 6 months post-surgery, ensuring the accuracy, completeness, and continuity of follow-up information.Follow-up personnel maintain close contact with patients through a combination of outpatient follow-up visits and phone calls, inquiring about and recording detailed core follow-up information: including the patient's psychological status, completing the secondary assessment of the HADS scale, recording scores and changes in classification; the patient's postoperative physical recovery, including daily status such as diet, sleep, and activity ability, as well as the presence of postoperative discomfort symptoms; the patient's laboratory test results and imaging examination results, simultaneously collecting blood routine, blood biochemistry, tumor markers, and imaging reports during the follow-up period; whether the patient has received anti-anxiety and depression medications, professional psychological intervention, or related treatments, recording the intervention methods and outcomes. Through standardized follow-up tracking, it is possible to dynamically observe the changes in anxiety and depression levels, the recovery process of physical function, and the stable state of tumor conditions, providing rich and systematic clinical data for in-depth analysis of the long-term association between tumor-derived protein expression levels and anxiety and depression in tumor patients, and exploring the clinical application value of tumor-derived proteins as molecular markers.

This study, from the precise determination of research subjects, the establishment of research foundations, in-depth analysis of clinical data, the scientific design and implementation of validation cohorts, to comprehensive and systematic observation indicators and follow-up plans, each link has been carefully designed and strictly implemented, aiming to conduct in-depth research on tumor-related anxiety and depression from the perspective of the correlation between tumor molecular expression and psychological phenotypes, clarifying the clinical value of tumor-derived proteins as molecular markers for tumor-related anxiety and depression, and providing a solid theoretical and practical basis for promoting the precision screening and individualized intervention of tumor-related anxiety and depression, with the potential to improve the comprehensive tumor treatment system and enhance the quality of life of tumor patients.

IV. Risk/Benefit Assessment

• Benefits Participation in this study may not benefit the patients. The tumor-derived protein targets focused on in this study have been confirmed by the research team's preliminary basic research to have the potential to become associated molecular markers for tumor-related anxiety and depression by mediating central nervous system inflammation to regulate emotional abnormalities. By validating its association with the level of anxiety and depression in tumor patients and its clinical application efficacy in clinical samples through this project, it is hoped to change the current clinical situation where there is a lack of specific molecular indicators for early screening of tumor-related anxiety and depression, providing new molecular evidence for the clinical precision identification and risk stratification of tumor psychological disorders. Based on the high incidence of tumor-related anxiety and depression in clinical practice and the current diagnostic and therapeutic difficulties with insufficient targeting of existing intervention measures, the research outcomes of this project are expected to improve the comprehensive tumor treatment system and provide a new direction for individualized intervention of psychological disorders in tumor patients, generating significant social and clinical application value.
• Risks The sample collection involved in this study includes collecting a small amount of tumor tissue samples during tumor surgery and peripheral venous blood draw. The risks of venous blood draw include temporary pain, discomfort, local bruising, and ecchymosis at the puncture site. Although the possibility of occurrence is extremely low, minor infection, slight bleeding, the formation of small hard knots due to local coagulation, or dizziness and fainting due to individual tolerance differences may still occur. The collection of tumor tissue samples is completed simultaneously with routine surgical operations and does not additionally increase surgical wounds or related surgical risks, without any special new traumatic risks. All sample collection procedures are strictly implemented by healthcare professionals with professional qualifications following standardized procedures, with thorough patient assessment before the procedure and the adoption of standardized protective and emergency measures during the procedure, which can minimize the probability of the occurrence of the aforementioned risks.

V. Data Management and Statistical Analysis Plan This project utilizes GraphPad Prism5 and SPSS software for statistical analysis and data analysis. All biological function experiments are repeated three or more times.Statistical differences between means were analyzed using the unpaired Student's t-test method, clinical sample data were analyzed using the Pearson χ2 test, and survival-related data were analyzed using Kaplan-Meier analysis and univariate COX regression analysis. Data are expressed as mean ± S.E.M. All tests were performed using two-tailed tests, and a P value < 0.05 was considered statistically significant.

VI. Data Confidentiality During the study, patient names, gender, and other personal information will be replaced with codes or numbers and strictly kept confidential, known only to relevant doctors. Patient privacy will be well protected. Research results may be published in journals, but no patient information will be disclosed. All medical records of the patients will be accessible to personnel of the research-initiating organization, relevant authorities, or an independent ethics committee to examine whether the study procedures are appropriate.

Inclusion criteria

• Patients with solid tumors aged ≥18 years and an expected survival period of ≥3 months;
• Patients who meet clinical diagnostic criteria and are pathologically/histologically confirmed as having solid tumors, including liver cancer patients (diagnosable by imaging);
• Patients who can complete standardized stratified assessments for tumor-related anxiety and depression, and are able to cooperate with researchers in completing psychological scales such as the Self-Rating Anxiety Scale (SAS) and the Self-Rating Depression Scale (SDS). They have not received any anti-anxiety/depression medications, professional psychological counseling, or psychiatric interventions before scale assessment;
• Patients with clear consciousness, normal language communication, comprehension, and cognitive abilities, without a history of psychiatric disorders, and who can independently provide feedback on research-related information and cooperate with follow-up;
• Patients who voluntarily participate in the study, fully understand the research objectives, procedures, potential risks, and benefits, and have signed a written informed consent form;
• Patients who can cooperate in completing the required laboratory tests (complete blood count, blood biochemistry, coagulation function, etc.) and clinical data collection before and during the perioperative period to ensure the completeness of research data.

Exclusion criteria

• Pregnant or lactating women;
• Patients aged <21 years at first visit and with an expected survival period of <3 months;
• Patients who have previously received anti-anxiety/depression medications or professional psychological interventions;
• Patients with a history or current diagnosis of psychiatric disorders, including schizophrenia, bipolar disorder, severe cognitive dysfunction, mental retardation, or those who cannot cooperate with SAS/SDS scale assessment or have contraindications to scale evaluation;
• Patients with severe organ dysfunction or severe underlying diseases, including: liver failure (Child-Pugh C grade), renal failure (serum creatinine >250 μmol/L or >2.83 mg/dL), New York Heart Association (NYHA) Class IV heart failure, active pulmonary tuberculosis, HIV infection, etc. (to be determined);
• Patients with contraindications to tumor tissue or blood sample collection, including coagulation disorders (INR >1.5, platelets <50×10⁹/L), severe bleeding tendency, or surgical specimens that cannot meet detection requirements (e.g., >50% necrotic tissue, insufficient tissue amount);
• Patients with emotional abnormalities due to non-tumor factors, including hyperthyroidism/hypothyroidism, severe malnutrition, chronic wasting diseases, or psychological stress disorders;
• Patients who have undergone non-tumor-related surgical procedures, enteral/parenteral nutrition support within the past 1 month, or have clinical symptoms such as gastrointestinal mechanical obstruction, intractable vomiting, ascites, significant edema, or large pleural effusion;
• Patients who are planned or currently using medications that may affect emotional assessment or protein expression detection during the study, including: long-term oral corticosteroids, 5-HT receptor agonists/antagonists (SSRIs, etc., even short-term use within 2 weeks before sampling is excluded), antipsychotic drugs, or gestagenic synthetic steroid derivatives (short-term inhaled/local use of steroids or intermittent use of inhaled bronchodilators are excluded);
• Patients with incomplete clinical data, inability to cooperate with research-related follow-up and testing, or those who refuse to sign the informed consent form or have poor compliance.