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.

Fig 1. Patient enrollment and sample collection for utDNA MRD analysis.

Patients with localized bladder cancer who were candidates for radical cystectomy were enrolled onto this study, as were healthy adult donors. Biofluid samples were then collected for utDNA analysis as shown in the schema. MRD, minimal residual disease; pCR, pathologic complete response; uCAPP-Seq, urine Cancer Personalized Profiling by Deep Sequencing; utDNA, urine tumor DNA.

Fig 2. uCAPP-Seq application of MRD gene panel to urine samples.

(A) Study schema. Urine and blood samples from 42 localized bladder cancer patients scheduled for radical cystectomy and 15 healthy adults were profiled by uCAPP-Seq. Samples from localized bladder cancer patients were collected preoperatively on the day of surgery. Sequencing libraries were prepared from urine cfDNA and peripheral blood germline DNA. utDNA analysis and results were primarily correlated with pathologic response in this study. (B) In silico application of the MRD panel, consisting of 49 consensus driver genes, to MIBC cases characterized by TCGA (n = 409) and DFCI/MSKCC (n = 50). A median of 5 mutations (silent and non-silent) per patient was detected in both datasets as indicated. (C) ROC curve classified pCR (n = 16) from no pCR (n = 26) patients by utDNA level, represented by the mutation with highest VAF. utDNA level derived from non-silent mutations (red curve) classified pathologic response significantly more accurately than silent mutations (blue curve). (D) Co-mutation plot showing non-silent driver mutations (with duplex support) detected in the urine of each patient with no pCR, patient with pCR, and healthy adult (bottom heatmap). utDNA level per variant is depicted within the heatmap, while the conglomerate level per subject is represented by the bar graph. Key patient characteristics are represented by the top heatmap. AUC, area under the curve; cfDNA, cell-free DNA; DFCI/MSKCC, Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center; MIBC, muscle-invasive bladder cancer; MRD, minimal residual disease; NGS, next-generation sequencing; pCR, pathologic complete response; ROC, receiver operating characteristic; Sn, sensitivity; Sp, specificity; TCGA, The Cancer Genome Atlas; uCAPP-Seq, urine Cancer Personalized Profiling by Deep Sequencing; utDNA, urine tumor DNA; VAF, variant allele fraction.

Fig 3. utDNA MRD analysis in patients with localized bladder cancer.

(A) Scatter plot of utDNA MRD levels (highest non-silent VAF per patient at preoperative time point) after square root transformation for 3 groups: healthy adults, patients with pCR, and patients with no pCR. The p-values were calculated using Mann–Whitney U test with α of 0.017 after Bonferroni correction. (B) ROC analysis classifying localized bladder cancer patients (either pCR or no pCR) versus healthy adults by utDNA level. AUC and Youden index–optimized sensitivity and specificity values are shown. (C) Stacked bar plots showing the proportions of each group with positive or negative utDNA MRD detection, determined using the optimal utDNA level threshold (VAF: 2.3%) that classified patients with no pCR from healthy adults. The p-values were calculated using Fisher exact test with α of 0.017 after Bonferroni correction. (D) Multivariate logistic regression predicting the absence of pCR. The heatmap shows logarithm of the OR for each variable in the logistic regression. Each bar shows a negative logarithm of the p-value, with the dashed line denoting α of 0.05. AUC, area under the curve; MRD, minimal residual disease; OR, odds ratio; pCR, pathologic complete response; ROC, receiver operating characteristic; Sn, sensitivity; Sp, specificity; Sqrt, square root; utDNA, urine tumor DNA; VAF, variant allele fraction; Y/N, yes/no.

Fig 4. Preoperative utDNA MRD as an early prognostic biomarker in localized bladder cancer patients.

(A) Kaplan–Meier analysis of PFS stratified by preoperative utDNA MRD detection and (B) pathologic response after radical cystectomy. utDNA MRD (+) patients had significantly worse PFS than utDNA MRD (−) patients (HR = 7.4; 95% CI: 1.4–38.9; p = 0.02). Patients with no pCR also had significantly worse PFS than patients with pCR (HR = 5.5; 95% CI: 1.1–28.1; p = 0.04). HRs and p-values were calculated using the Mantel–Haenszel and Mantel–Cox tests, respectively. (C) Serial urine cfDNA analysis for MIBC patient BC-1050. Imaging after neoadjuvant chemotherapy was equivocal with residual bladder wall thickening. utDNA levels increased during neoadjuvant chemotherapy with a COSMIC- and OncoKB-annotated nonsynonymous ERCC2 mutation having the highest VAF. utDNA levels then decreased and became undetectable at the preoperative MRD time point, concordant with pCR on surgical pathology. The patient remained disease free on long-term follow-up. (D) Serial urine cfDNA analysis for MIBC patient BC-1045. Imaging after neoadjuvant chemotherapy noted decreased but persistent bladder wall thickening. utDNA levels decreased during neoadjuvant chemotherapy, with a KMT2D stop–gain mutation having the highest VAF. utDNA levels then increased at the preoperative MRD time point with evidence of 2 new emergent nonsynonymous mutations in ERBB2 and ARID1A, not present earlier. Consistent with this positive utDNA MRD result, the patient had pathological nodal involvement despite achieving a pCR in the urothelium, and, less than a year later, developed progressive disease that prompted the start of treatment with atezolizumab. cfDNA, cell-free DNA; CI, confidence interval; COSMIC, Catalogue of Somatic Mutations in Cancer; HR, hazard ratio; MIBC, muscle-invasive bladder cancer; MRD, minimal residual disease; pCR, pathologic complete response; PFS, progression-free survival; utDNA, urine tumor DNA; VAF, variant allele fraction.

Fig 5. utDNA MRD as a biomarker for adjuvant treatment personalization.

(A) Comparison of non-silent mutational load detected by whole exome sequencing and uCAPP-Seq for MIBC cases in TCGA (n = 409), using an expanded gene panel for assessing TMB (referred to as the TMB panel; S3 Table). Linear regression was performed (Pearson’s r = 0.84; equation shown) to interpolate exome-wide TMB based on the number of non-silent mutations detected by uCAPP-Seq. (B) Scatter plot depicting inferred TMB among patients with positive utDNA MRD in our cohort with the median indicated by a horizontal black line. (C) Potential adjuvant treatment strategies for utDNA MRD (+) patients based on urine-inferred TMB. (D) Vignette depicting MIBC patient BC-1171 who was utDNA MRD (+) at the preoperative time point, concordant with no pCR on surgical pathology with evidence of locally advanced pT3a disease (AJCC 8). Six non-silent mutations were detected in preoperative urine from this patient as indicated. Urine-inferred TMB was elevated at 204. This patient was randomized to pembrolizumab on a clinical trial and has shown no evidence of disease on long-term follow-up, consistent with our prediction for a patient with high inferred TMB. AJCC 8, American Joint Committee on Cancer 8th edition; MIBC, muscle-invasive bladder cancer; MRD, minimal residual disease; pCR, pathologic complete response; TCGA, The Cancer Genome Atlas; TMB, tumor mutational burden; TURBT, transurethral resection of bladder tumor; tx, treatment; uCAPP-Seq, urine Cancer Personalized Profiling by Deep Sequencing; utDNA, urine tumor DNA.