Identification of Apo-A1 as a biomarker for early diagnosis of bladder transitional cell carcinoma

Abstract

Background: Bladder transitional cell carcinoma (BTCC) is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis. Availability of urinary tumor biomarkers represents a convenient alternative for early detection and disease surveillance because of its direct contact with the tumor and sample accessibility.

Results: We tested urine samples from healthy volunteers and patients with low malignant or aggressive BTCC to identify potential biomarkers for early detection of BTCC by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) and bioinformatics analysis. We observed increased expression of five proteins, including fibrinogen (Fb), lactate dehydrogenase B (LDHB), apolipoprotein-A1 (Apo-A1), clusterin (CLU) and haptoglobin (Hp), which were increased in urine samples of patients with low malignant or aggressive bladder cancer. Further analysis of urine samples of aggressive BTCC showed significant increase in Apo-A1 expression compared to low malignant BTCC. Apo-A1 level was measured quantitatively using enzyme-linked immunosorbent assay (ELISA) and was suggested to provide diagnostic utility to distinguish patients with bladder cancer from controls at 18.22 ng/ml, and distinguish patients with low malignant BTCC from patients with aggressive BTCC in two-tie grading system at 29.86 ng/ml respectively. Further validation assay showed that Apo-A1 could be used as a biomarker to diagnosis BTCC with a sensitivity and specificity of 91.6% and 85.7% respectively, and classify BTCC in two-tie grading system with a sensitivity and specificity of 83.7% and 89.7% respectively.

Conclusion: Taken together, our findings suggest Apo-A1 could be a potential biomarker related with early diagnosis and classification in two-tie grading system for bladder cancer.

Figure 1

2-DE profiles of urine samples. Pooled urine samples from 24 healthy individuals (A), 24 patients with low malignant BTCC (B) and 16 patients with aggressive BTCC (C) were resolved on 2-DE gels. Protein spots were visualized using CBB. Spots with more than 10-fold difference of two matched spots from two different groups were numbered. The red arrows labeled with numbers indicate the position of the differentially expressed proteins.

Figure 2

Western blot validation of the proteins identified by 2-DE coupled with MS. Fb, LDHB, Apo-A1, CLU and Hp expression in pooled urine samples from healthy individuals, patients with low malignant or aggressive BTCC were further detected by Western blot. Equal protein amount (25 μg) from each group was loaded onto 10% SDS-PAGE gels. Antibodies were accepted as detecting a single predominant band at the expected molecular weights.

Figure 3

2-DE profiles of Apo-A1 expression on independent series of urine samples. Independent urine samples from healthy individuals, patients with bladder benign damages, patients with low malignant or aggressive BTCC were resolved on 2-DE gels and stained by sliver. Differentially expressed Apo-A1 was highlighted with an open oval which was further identified by MALDI-TOF/TOF-MS. Three representative gels (Sample I, II and III) of each group were shown.

Figure 4

Apo-A1 as a biomarker for predicting and classifying BTCC. (A) Quantitative proteomic analysis of Apo-A1 in urine samples by ELISA. *P < 0.01, Student's t-test. (B) Urianry Apo-A1 distinguishes bladder cancer on independent series of urine samples of patients with BTCC and controls. ROC curve of urinary Apo-A1 as a detection marker for bladder cancer was based on a series of 72 urine samples. Among these, 40 had cancer-positive. The optimal cutoff was 18.22 ng/ml, and the AUC obtained was 0.928 (95% CI 0.870-0.986). (C) Urinary Apo-A1 classifies with a different series of urine samples of patients with low malignant or aggressive BTCC. ROC curve of urinary Apo-A1 as a detective classification marker for BTCC was based on a series of 40 urinary specimens, which consists of 24 low malignant BTCC and 16 aggressive BTCC. The optimal cutoff was 29.86 ng/ml, and the AUC obtained was 0.875 (95% CI 0.758-0.992).