Genomic profile of urine has high diagnostic sensitivity compared to cytology in non-invasive urothelial bladder cancer

Abstract

Cytology is widely conducted for diagnosis of urothelial bladder cancer; however, its sensitivity is still low. Recent studies show that liquid biopsies can reflect tumor genomic profiles. We aim to investigate whether plasma or urine is more suitable for detecting tumor-derived DNA in patients with early-stage urothelial bladder cancer. Targeted sequencing of 71 genes was carried out using a total of 150 samples including primary tumor, urine supernatant, urine precipitation, plasma and buffy coat from 25 patients with bladder cancer and five patients with cystitis and benign tumor. We compared mutation profiles between each sample, identified tumor-identical mutations and compared tumor diagnostic sensitivities between urine and conventional cytology. We identified a total of 168 somatic mutations in primary tumor. In liquid biopsies, tumor-identical mutations were found at 53% (89/168) in urine supernatant, 48% (81/168) in urine precipitation and 2% (3/168) in plasma. The high variant allele fraction of urine was significantly related to worse clinical indicators such as tumor invasion and cytological examination. Although conventional cytology detected tumor cells in only 22% of non-invasive tumor, tumor diagnostic sensitivity increased to 67% and 78% using urine supernatant and precipitation, respectively. Urine is an ideal liquid biopsy for detecting tumor-derived DNA and more precisely reflects tumor mutational profiles than plasma. Genomic analysis of urine is clinically useful for diagnosis of superficial bladder cancer at early stage.

Keywords: NGS; diagnosis; liquid biopsy; urine; urothelial bladder cancer.

Figure 1

Scheme of sample preparation. Before transurethral resection of bladder tumor (TURBT), five different samples were obtained from each of the 25 patients with urothelial BC: buffy coat, plasma, urine precipitate (ppt), urine supernatant (sup), and primary tumor tissues. Urine cytology specimens were also prepared. DNA was extracted from these samples and analyzed by targeted sequencing

Figure 2

Mutation profiles identified in primary tumor, urine supernatant, urine precipitate, and plasma. Heat maps show identical mutations in the indicated samples corresponding with primary tumor mutations. Variant allele fraction values are shown in blue (high value) and light blue (low value) boxes. Grey boxes indicate no identified mutation in samples. ppt, urine precipitate; sup, urine supernatant

Figure 3

Variant allele fractions (VAF) of identified mutations in urine supernatant and urine precipitate are related to clinical indicators. Box plots show the VAF of identical mutations corresponding to tumor in urine (A‐D). High VAF mutations were detected in invasive tumors compared to non‐invasive tumors in urine supernatant (sup) (A) and precipitate (ppt) (B).High VAF mutations were observed in class V tumor determined by cytological examination compared to class I‐III tumor in urine supernatant (C) and precipitate (D). P‐value was calculated with Student's t test

Figure 4

Tumor diagnostic sensitivity of conventional cytology and genetic analyses of urine and plasma samples. Comparison of the tumor diagnostic rate in each patient. Clinical information (gender, invasion, tumor stage, cytology, tumor grade) is shown at the top of the figure. Conventional cytology was used to classify tumors as class I‐V. Class I, II and III indicates tumor negative (light pink) and class V indicates tumor positive (pink). Brown boxes indicate positive identical mutations corresponding to the primary tumor in urine supernatant (sup), urine precipitate (ppt), and plasma. No mutations are indicated by grey boxes. I, invasive tumor; NI, non‐invasive tumor

Figure 5

High diagnostic sensitivity of urine genomic profiles. Tumor diagnostic sensitivity of urine was higher than that of conventional cytology. Significant difference was observed in all tumor (n = 25) or non‐invasive tumor (n = 18), but not in invasive tumor (n = 7). P‐value was calculated with Pearson's chi‐squared test. ns, not significant. ppt, urine precipitate; sup, urine supernatant