Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study

Abstract

Background The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. Methods and findings We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD–positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4–38.9; p = 0.02) compared to utDNA MRD–negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD–positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point. Conclusions utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.

Disease Management of Clinical Complete Responders to Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer: A Review of Literature

“…Urinary cytology plays a significant role in the detection and surveillance of bladder cancer, and detection of soluble proteins and omics biomarkers have been further explored, such as the detection of nuclear matrix proteins and tests of DNA methylation ( 57 , 58 ). Recently, liquid biopsy based on urine tumor DNA has also been demonstrated to be a highly sensitive technique in the detection of minimal residual disease (MRD) ( 59 ). These tests could be reliable complements to urinary cytology with high sensitivity.…”

ctDNA: An emerging neoadjuvant biomarker in resectable solid tumors

“…Like the application of ctDNA in LARC, Chauhan and colleagues asked whether evaluation of urinary ctDNA (utDNA) could differentiate pCR from non-pCR in patients being treated with neoadjuvant therapy for MIBC [ 1 ]. Development of an alternative to pCR in this setting could avoid surgical cystectomy and urinary diversion in excellent prognosis patients.…”

“…Three studies presented within this special issue of PLOS Medicine focus on evaluation of circulating tumor DNA (ctDNA) as a response biomarker in early-stage solid tumours. Both Yaqi Wang and Pradeep Chauhan and their respective colleagues evaluate ctDNA as a tool capable of predicting complete pathological response (pCR) in locally advanced rectal cancer (LARC) and muscle invasive bladder cancer (MIBC), respectively [ 1 , 2 ]. Jeanne Tie and colleagues focus on ctDNA evaluation in high-risk metastatic colorectal cancer with liver metastases (CRLM), both during neoadjuvant therapy and following surgery and adjuvant therapy [ 3 ].…”

Liquid Biopsy Biomarkers for Immunotherapy in Non-Small Cell Lung Carcinoma: Lessons Learned and the Road Ahead

“…New studies have tried to avoid this issue by turning to other more readily available fluids, such as urine. In bladder cancer patients, for example, Chauhan et al successfully calculated TMB from urine ctDNA [84]. Translating this type of study to immunotherapy in NSCLC patients could be of interest to therefore avoid the present limitations.…”