This study is a prospective cohort study of the anxiety levels of cancer pain patients with different OPRM1 genotypes that are conducted at a single institution. Our study was approved by the Yunfu People's Hospital Medical Ethics Committee (2022A050) and was conducted in compliance with the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects. The corresponding authors designed, managed, and provided financial support for the study. All data collection occurred prospectively. Clinical data were registered by physicians. Data analysis was conducted by first authors under the guidance of a statistician. The eligibility criteria: (1) Participants aged over 18 years; (2) Cancer patients requiring appropriate opioid analgesics for pain relief; (3) Individuals with good compliance who can effectively return for follow-up visits; (4) non-cancer pain patients. Key exclusion criteria included: (1) Participants under 18 years of age; (2) Patients with non-cancer pain; (3) Patients suffering from severe mental disorders that might interfere with the normal conduct of the study; (4) Individuals with incomplete data; (5) Participants with poor compliance; (6) Individuals without complete and valid OPRM1 gene testing results. Informed consent was obtained from all individual participants included in the study.

Prior to enrollment, all patients completed comprehensive pretreatment assessments to identify any opioid contraindications. Between January, 2023 to May, 2024, 76 cancer pain patients who met the inclusion criteria and were receiving opioid treatment participated in this study. All recruited patients were from Yunfu People's Hospital. The diagram is shown in (Figure 1).

All patients completed relevant questionnaires before receiving opioid treatment.

Each patient's anxiety status was assessed using the Self-Rating Anxiety Scale (SAS). The total score was calculated by summing the ratings of all items. A total score above 36 points was considered a positive screen, indicating the possible presence of anxiety symptoms requiring further examination. The higher the score, the more severe the anxiety.

The European Organization for Research and Treatment of Cancer (EORTC) developed the Quality of Life Questionnaire Core 30 (QLQ-C30) to assess the quality of life in cancer pain patients (13). This questionnaire includes 30 items covering multiple domains of quality of life, such as overall health status, functional scales and symptom scales.

Each patient's depression status was evaluated using the Self-Rating Depression Scale (SDS). Items are rated on a 4-point scale (1–4 for negative statements, 4-1 for positive statements), with scores summed across all 20 items, multiplied by 1.25, and rounded to the nearest whole number to derive the standard total score. Scores below 50 indicate normal, 50-59 mild depression, 60-69 moderate depression, and 70-79 severe depression.

Nutritional status was assessed using a modified Subjective Global Assessment (SGA) with five grades: Normal (no weight loss, adequate intake), Mild malnutrition (5%–7.5% weight loss, slight dietary reduction), Moderate malnutrition (7.5%–10% weight loss, functional impairment), Severe malnutrition (>10% weight loss, marked depletion), and Overweight (BMI≥25 without malnutrition). Evaluations included weight history, dietary intake, and physical examination for muscle/fat loss.

All patients underwent pain assessment using the Numeric Rating Scale (NRS; 0–10 point scale) both before initiation and after completion of opioid therapy to evaluate treatment-related changes in pain status. All patients underwent an NRS assessment for pain before receiving opioid treatment. After administering an equivalent dose of opioids and once the patients experienced pain relief, another NRS assessment was conducted. The dose of opioid medication required for pain relief was recorded for each patient.

Oral mucosal cell samples were collected from the buccal walls of all participants using disposable sampling swabs and reserved for subsequent use. Swabs that could not be analyzed immediately should be stored at 2–8 °C and should not exceed 7 days in storage. After collecting, the swabs were allowed to thaw in the reagent room, followed by instantaneous centrifugation and reserved for further processing. A volume of 400 μL of sample extraction solution (CQ-ENH type) was pipetted into a 1.5 mL centrifuge tube, into which the swab head was immersed, and the stem of the swab was broken off so that the swab head was submerged in the extraction solution. The mixture was vortexed to ensure homogeneity (allowed to stand for 1 min). Subsequently, 1.0 μL of the processed sample was spotted onto the wall of the reagent tube, followed by centrifugation before being loaded onto the detection instrument. According to the manufacturer's instructions, in situ hybridization (ISH) was used to analyze the OPRM1 rs1799971 (A118G) gene polymorphism (reagents provided by Jinan Guangyin Medical Technology Co., Ltd.). The FISH method employed in this study is a liquid-phase hybridization system specifically designed for SNP genotyping of extracted DNA, rather than for traditional tissue localization applications. This method has undergone consistency validation using blood and oral exfoliated cell samples, with validation results demonstrating 100% genotypic concordance. The method is reliable and suitable for genotyping analysis of oral swab samples. Full validation data are provided in Attachment 1.

In the course of our study, various types of opioid medications were utilized, making it necessary to convert to equivalent doses when comparing patients to different medications. We conducted a comparative analysis of the equivalent analgesic doses of several common analgesics and have listed the dose conversion ratios for these drugs administered via intramuscular (IM) and oral (PO) routes in. Based on the total daily oral morphine consumption, we converted to the recommended dosage of fentanyl patches. These dose conversion relationships are derived from publications by the American Society of Health-System Pharmacists (ASHP) and the instructions for use accompanying the fentanyl transdermal patch (Duragesic) (38).

The primary objective of this study is to investigate the potential association between the OPRM1 gene and anxiety in a cohort of cancer pain patients. Secondary objectives include examining the correlation between the OPRM1 gene and the dose of opioid medication used.

Quantitative data are presented as median (interquartile range) or mean ± standard deviation, and categorical data are presented as number of cases (n, %). No missing data was encountered in the dataset utilized for this analysis. The relative frequencies of each genotype and allele were calculated and compared using chi-square analysis. Statistical comparisons of SAS, SDS, and QLQ-C30 scores among chronic cancer pain patients were performed using the “CreateTableOne()” function from the “tableone” R package, which automatically selects appropriate tests: t-tests or Wilcoxon rank-sum tests for continuous variables based on normality distribution, and Pearson's chi-square tests (with automatic switch to Fisher's exact test when expected cell counts <5) for categorical variables. To investigate factors associated with anxiety in cancer patients with pain, logistic regression analysis was employed. Variables with a significance level of p < 0.05 were included in the subsequent multivariate analysis. For categorical variables with multiple levels, the entire variable was retained in multivariate analysis if any level reached statistical significance (p < 0.05), maintaining the integrity of the categorical structure. Multicollinearity among independent variables was evaluated using variance inflation factors (VIFs). Model fit was assessed using a likelihood ratio test.

A P-value < 0.05 was considered statistically significant. Statistical analyses were carried out with R software package version 4.4.0 and SPSS 27.0.

The main clinical and demographic characteristics of patients are presented in (Table 1).

The population was predominantly male (80.26%) with a median age of 64 years (IQR:54–70). Anthropometric measurements revealed a median height of 1.65 m, weight of 54.00 kg, and BMI of 20.07 kg/m². Smoking history was reported in 23.68% of patients, while no cases of drug abuse were documented. Regarding cancer distribution showed head and neck tumors as most prevalent (26.32%), followed by lung (19.74%) and colon cancers (14.47%). Pain locations were primarily abdominal (31.58%) and cephalic (27.63%). Current antitumor therapy was administered to 73.68% of patients. The psychological evaluation revealed that 31 patients (40.79%) presented with anxiety. Regarding depressive symptoms, 17 cases (22.37%) were classified as mild, 9 (11.84%) as moderate, and 2 (2.63%) as severe. Nutritional status evaluation demonstrated comparable proportions of normal nutrition (26.32%) and severe malnutrition (26.32%). The cohort exhibited allele frequencies of 0.560 (A) and 0.440 (G) for the studied genetic variant.

The morphine dosage required by OPRM1 genotype patients and their NRS scores are presented in (Table 2). A comparative analysis of equianalgesic dosing was conducted, with intramuscular and oral dose conversion ratios provided in Supplementary Table S1 and fentanyl patch dosing recommendations based on daily oral morphine intake summarized in Supplementary Table S2.

In this study, the OPRM1 genotyping of 76 pain patients revealed 24 cases (31.6%) with the AA genotype, 37 cases (48.7%) with the AG genotype, and 15 cases (19.7%) with the GG genotype. Statistical analysis confirmed that the genotype distribution was in accordance with Hardy-Weinberg equilibrium, indicating that the sample population was representative. We initially analyzed whether there were differences in pain and opioid dosage requirements among cancer pain patients with different genotypes. The distribution of opioids used in this population is as follows: oxycodone (n = 53, 69.74%), tramadol (n = 8, 10.53%), fentanyl (n = 6, 7.89%), morphine (n = 5, 6.58%), and combination therapy (n = 4, 5.26%). The results showed no statistically significant differences among the different genotype groups in terms of daily morphine dosage (p = 0.911), NRS scores before and after treatment (p = 0.402, p = 0.999), ΔNRS scores (p = 0.875), and the Average Dose of morphine (p = 0.916).

The scores for each dimension and the statistical results are presented in (Table 3).

The study results indicate that there are no significant differences between patients with the A/A genotype and the combined G allele carriers in multiple dimensions of the QLQ-C30 and the SDS scores. However, a significant difference was observed in anxiety scores between genotype groups (p = 0.017). Patients with the A/A genotype demonstrated significantly higher median anxiety scores (36.00 [IQR: 28.00–42.00) compared to those with A/G + G/G genotypes (27.00 [IQR: 22.00–36.25).

To identify key determinants of anxiety status, univariate analysis revealed significant associations with genotype, pain location, antitumor therapy, breakthrough pain, pretreatment NRS scores, and depression. Notably, multivariate analysis confirmed that G allele carriers (OR:0.15, 95%CI: 0.02–0.91, P = 0.040) and antitumor therapy (OR:0.10, 95%CI: 0.01–0.77, P = 0.027) served as independent protective factors, while breakthrough pain (OR:6.65, 95%CI: 1.00–44.16, P = 0.050), higher pretreatment NRS scores (OR:2.80, 95%CI: 1.05–7.44, P = 0.039), and depression (OR:61.03, 95%CI: 8.40–443.66, P < 0.001) emerged as independent risk factors for anxiety (Table 4). Most VIFs were below 5, with the exception of “AST after treatment” which had a VIF of 6.25 (Supplementary Table S3). The full model provided a significantly better fit to the data than the null model, as evidenced by a likelihood ratio test (χ²= 75.33, p < 0.001).