Study population
The data of 118 patients who presented to the Mersin University Urology Clinic between June 2022 and December 2022 who were followed and treated in our clinic for NMIBC and who received intravesical BCG treatment when necessary, as well as 56 individuals without any malignancies, were prospectively examined. Individuals with invasive and/or metastatic bladder cancer, those under 18 years of age, those with NMIBC who did not receive intravesical BCG treatment, those with malignancies other than bladder cancer, and those who did not sign informed consent forms were excluded from the study. Patients with other malignancies were excluded from the study to ensure that the results were not influenced, as the fibronectin molecule or fibronectin gene polymorphisms may play a role in the formation of various malignancies. Among a total of 118 NMIBC patients, 73 underwent intravesical BCG treatment once a week for a minimum of 6 weeks, starting two weeks after the initial diagnosis. Patients who completed at least the induction phase of intravesical BCG therapy, along with 56 healthy individuals without any malignancy, were included in the study. All individuals’ demographic data, such as age, weight, height, body mass index (BMI), family history, and smoking status, were examined. Informed consent forms were signed by all individuals included in the study. Ethical approval for the research was obtained from the Mersin University Faculty of Medicine Local Ethics Committee with the decision dated 23.03.2022, numbered 2022/200. Informed consent forms were signed by all individuals included in the study.
Sample collection and storage
The analysis of serum FN levels and the detection of the FN gene polymorphisms RS 10,202,709 and RS 35,343,655 were conducted at the Mersin University Medical Biochemistry Laboratory. Blood samples were collected from patients and healthy individuals and put into 2 ml EDTA tubes for polymorphism analysis. For enzyme-linked immunosorbent assay (ELISA), blood samples were collected into 5 ml plain biochemistry tubes. The blood samples collected into simple biochemistry tubes were centrifuged at 4000 rpm for 10 min, and the sera were separated. Storing serum samples at -80 °C slows down protein degradation, thereby preserving the long-term stability of biomolecules like fibronectin, which enhances the reliability of research results. Therefore, the serum samples were stored at -80 °C until the analysis day. The blood samples collected in EDTA tubes were stored at + 4 °C.
Measurement of fibronectin levels
Serum FN levels were determined using the ELISA method, following the protocol recommended by the manufacturer. This was carried out using an ELISA washer and ELISA reader (Thermo Scientific) devices. For each analysis, the concentrations were calculated for each sample using the optical density values corresponding to known standards by utilizing the curves and equations drawn.
DNA isolation and polymorphism analysis
DNA isolation was carried out from the blood samples stored at + 4 °C using a DNA isolation kit (Roche Diagnostics, Mannheim, Germany). The FN gene polymorphisms RS 10,202,709 and RS 35,343,655 were detected in the isolated DNA samples using a real-time PCR (polymerase chain reaction) device (Roche LightCycler 480; Roche Diagnostics, Mannheim, Germany). The reaction mixtures were prepared according to the protocol specified by the manufacturer, and the PCR conditions listed in Supplementary Table 1 were applied.
Statistical
In this study, continuous measurements were assessed for normality using the Shapiro-Wilk test. This test was selected due to its robustness and widespread use in determining whether a continuous variable adheres to a normal distribution, a critical assumption for many parametric statistical analyses. The hypothesis tested here was that the continuous variables follow a normal distribution, which is essential for the validity of subsequent parametric tests.
For comparing constant measurements between patient and control groups, Student’ s t-test was employed. This test is appropriate for evaluating the means of two independent groups when the data is continuous and normally distributed. The hypothesis tested was that there is no significant difference in the means of the continuous variables between the patient and control groups.
Descriptive statistics, including means and standard deviations, were reported to summarize the central tendency and variability of continuous variables.
To analyze differences in categorical variables between groups, Pearson’ s chi-squared test, Fisher’ s exact chi-squared test, and the likelihood ratio chi-squared test were utilized. Pearson’ s chi-squared test was applied for larger sample sizes, while Fisher’ s exact test was used for small sample sizes or sparse data. The likelihood ratio chi-squared test served as an alternative that may offer more reliability in specific situations. The hypothesis tested was that there is no association between categorical variables and group membership.
Counts and percentages were provided as descriptive statistics for categorical variables to offer a clear understanding of the distribution of these variables across groups.
Binary logistic regression analysis was conducted to identify potential risk factors influencing bladder cancer incidence. This method is suitable for modeling the relationship between multiple independent variables and a binary outcome, such as the presence or absence of bladder cancer. The hypothesis tested was that certain independent variables are significant predictors of bladder cancer incidence.
Hardy-Weinberg equilibrium analysis was performed to verify whether gene polymorphisms were in equilibrium within the population, an essential consideration for genetic association studies to ensure the sample’ s representativeness of the general population. The hypothesis tested was that the observed genotype frequencies do not deviate from those expected under Hardy-Weinberg equilibrium.
Counts and percentages were also presented as descriptive statistics to provide a comprehensive overview of the data.
A paired samples t-test was used to evaluate differences between early and late recurrence groups. This test is appropriate for comparing two related samples within the same group of patients, allowing for the assessment of changes over time or between conditions. The hypothesis tested was that there is no significant difference in the means of the continuous variables between the early and late recurrence groups.
Furthermore, a one-way ANOVA test was employed to examine genotype differences in gene polymorphisms concerning continuous measurements. One-way ANOVA is suitable for comparing means across three or more groups, enabling the detection of overall differences between genotypes. The hypothesis tested was that there are no significant differences in the means of continuous variables across different genotypes.
Levene’ s test was conducted to assess the homogeneity of variances, ensuring the validity of tests such as ANOVA and t-tests. The hypothesis tested was that the variances are equal across groups.
A significance level of p < 0.05 was considered statistically significant throughout the analyses, indicating that the probability of observing the results by chance is less than 5%.
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