Accumulating evidence implies the utility of DNA-based urine biomarkers for initial detection of bladder cancer (BC) and surveillance of non-muscle-invasive BC [1, 2]. We have previously described gene panels with utility for these indications, identifying UBC-associated mutations in 96% of all BCs, such that the associated urine test is not reliant on the initial identification of mutations in primary tumour tissue [1]. By contrast, the utility of urine as a liquid biopsy for the surveillance of patients with muscle-invasive BC (MIBC) treated by bladder preservation (radiotherapy ± chemotherapy) remains understudied; one previous publication describes microsatellite analysis of urinary DNA to detect bladder recurrences in five out of six radiotherapy patients [3].

We undertook a pilot study to evaluate whether measuring common BC-associated mutations in urinary DNA can contribute to the monitoring of treatment responses in patients with organ-confined MIBC treated with curative intent. Our objectives were to: (i) investigate the potential of urine DNA analysis before, during and after treatment as indicators of treatment response; (ii) investigate the prognostic value of an absence of detectable genomic alterations post treatment; and (iii) compare two orthogonal methodologies for detecting variants in urinary DNA (capture and Illumina sequencing vs PCR and Ion Torrent sequencing).

As part of the TUXEDO trial (a phase I/II feasibility study of cetuximab plus 5FU and mitomycin C or cisplatin with concomitant radiotherapy in patients with organ-confined MIBC; ethics approval 11/LO/1313, protocol at https://www.birmingham.ac.uk/research/crctu/trials/tuxedo/index.aspx), urine samples were collected from patients with MIBC before, during and after treatment with chemoradiotherapy. Briefly, urine samples (50 mL) were collected prior to treatment on the first day of weeks −1 to +7 (treatment completion) and at one post-treatment visit using urine preservation tubes (Norgenbiotek.com). Urine samples were centrifuged for 10 min at 2000g and DNA extracted from the pellet using the QuickDNA kit (Zymoresearch.com) and quantitated by Qubit. Capture-based libraries were prepared using 25 ng urine cell pellett DNA (cpDNA) and Nonacus Cell3Target (Nonacus.com). Target enrichment was via a custom panel covering c.10 kb and including hotspots/regions of 29 genes: coding regions of 23 genes plus the TERT promotor and an additional five non-coding mutation hotspots, as previously defined by large-scale tumour sequencing [1, 4]. Libraries were sequenced on an Illumina Nextseq system (2 × 150 bp); reads were aligned to hg19 genome version using BWA. Variant allele frequencies (VAFs) were extracted using the bam-readcount tool for the disease-associated single-nucleotide variants (SNVs) identified previously [1, 4]. We considered a 0.5% VAF to be the limit of detection for SNVs [5], and a 1% VAF to be the limit of quantitation. We report quantitative changes in the VAFs of urinary SNVs during treatment for all SNVs that exceeded 1% VAF at any time in each patient. Average raw and consensus read depths were 27 100× and 3000×, respectively.

Multiplex-PCR-based libraries were prepared using AmpliseqHD reagents (ThermoFisher) and workflow. Two panels of target-specific primers and 2 × 20 ng urine cpDNA were used to amplify the same target regions as for the capture-based approach. The libraries were sequenced using 540 chips on an S5 Ion Torrent system (2 × 100 bp; ThermoFisher). Alignments, consensus-building and variant calling were performed using Ion Reporter software. Average raw and consensus read depths were 79 300× and 5500×, respectively. We excluded AmpliseqHD data for the TERT promoter as this GC-rich amplicon did not give sufficient consensus reads for reliable variant calling.

In this way, common UBC-associated mutations were determined longitudinally in six patients. For one patient, no mutations were detected at any time point. For coding and TERT promoter mutations, cpDNA of four out of five patients contained high levels of tumour DNA (VAFs 4–20%) at baseline (post-transurethral resection, pre-chemoradiotherapy). VAFs in cpDNA decreased to lower levels in all patients by the end of treatment (Fig. 1).

In patient A, multiple mutations in the TERT promoter were detected at >20% VAF at baseline. The unusual co-occurrence of the 242/243 and 250 TERT mutations was confirmed by Sanger sequencing (data not shown). These mutations were detected at higher VAFs after 1 week of cetuximab-loading and after 1 week of radiotherapy, then decreased rapidly to approximately 5% VAF, remained clearly detectable through the later stages of treatment, and were present at 1.4% VAF after treatment completion. Patient A relapsed with local recurrence (grade/stage unknown) 7 months later.

In patient B, TERT 228A and FGFR3 S249C mutations were present at baseline; both were undetectable post-treatment. Patient B remained disease-free 16 months post-treatment.

In patient C, TERT and TP53 mutations were present at baseline and, despite showing a downward trend, remained detectable at 1.7% VAF after treatment. Patient C was diagnosed with malignant ascites 3 months post-treatment.

In patient D, TERT and TP53 mutations were present at baseline, dropped rapidly during treatment, and were undetectable after completion of treatment. Patient D was diagnosed with local recurrence (G3pT1) 5 months post-treatment.

In patient E, a TERT promoter mutation was present at low VAF at baseline; this mutation did not show a clear trend over time and remained detectable at most time points. Patient E was diagnosed with local recurrence (G3pT1) 9 months post-treatment.

PCR-based library preparation (AmpliseqHD) combined with Ion Torrent sequencing verified Illumina-based mutation detection and quantitation in cpDNA, except for the TERT promoter which amplified poorly (data not shown). VAFs measured by the two methods correlated well (r² = 0.96), and AmpliseqHD confirmed 84%, 94% and 98% of SNVs detected by capture-based sequencing at ≥0.5%, 1% and 2% VAF, respectively. Copy number profiles (a by-product of off-target reads from capture-based target enrichment) for all cpDNA were inspected manually; copy number variant levels mirrored SNV levels (data not shown).

In summary, two out of the four patients who relapsed (three local, one distant, 3–9 months after completing treatment) had undetectable urinary VAFs on treatment completion. This finding is particularly surprising for the two out of three bladder recurrences with undetectable urinary VAFs on treatment completion given that, in other cancer settings, the ‘clearance of mutations’ in liquid biopsy samples is associated with significantly improved outcomes [6]. The corollary is that two out of the three patients with detectable mutations on treatment completion experienced relapse within 7 months. Notwithstanding, we suggest that urine-based liquid biopsy monitoring of post-radiotherapy MIBC patients remains challenging, and should be combined with plasma ctDNA monitoring [7], or primary tumour tissue sequencing (to permit personalized urinary liquid biopsies with much lower detection thresholds [8]), or both. Methodologically, both targeted capture-based and PCR-based library preparation and next-generation sequencing can be used to identify common BC-associated mutations in urinary cpDNA. These pilot data suggest the need for further liquid biopsy research in this specific MIBC setting.

越来越多的证据表明基于 DNA 的尿液生物标志物可用于膀胱癌 (BC) 的初步检测和非肌肉侵袭性 BC 的监测 [ 1, 2 ]。我们之前已经描述了可用于这些适应症的基因组，在 96% 的所有 BC 中鉴定出 UBC 相关突变，因此相关的尿检不依赖于原发性肿瘤组织中突变的初步鉴定 [ 1 ]。相比之下，尿液作为液体活检监测接受膀胱保留（放疗±化疗）治疗的肌肉浸润性 BC (MIBC) 患者的效用仍未得到充分研究。之前的一份出版物描述了尿液 DNA 的微卫星分析，以检测六分之五的放射治疗患者的膀胱复发 [ 3]。

我们进行了一项初步研究，以评估测量尿 DNA 中常见的 BC 相关突变是否有助于监测以治愈为目的的器官局限型 MIBC 患者的治疗反应。我们的目标是： (i) 研究治疗前、治疗中和治疗后尿液 DNA 分析作为治疗反应指标的潜力；(ii) 研究治疗后没有可检测到的基因组改变的预后价值；(iii) 比较两种用于检测尿液 DNA 变异的正交方法（捕获和 Illumina 测序与 PCR 和 Ion Torrent 测序）。

作为 TUXEDO 试验的一部分（西妥昔单抗加 5FU 和丝裂霉素 C 或顺铂联合放疗对器官受限 MIBC 患者的 I/II 期可行性研究；伦理批准 11/LO/1313，协议在 https://www. birmingham.ac.uk/research/crctu/trials/tuxedo/index.aspx），在放化疗治疗之前、期间和之后从 MIBC 患者收集尿液样本。简而言之，在第-1至+7周的第一天（治疗完成）治疗前和治疗后一次使用尿液保存管（Norgenbiotek.com）收集尿样（50 mL）。将尿液样品以 2000 g离心 10 分钟使用 QuickDNA 试剂盒 (Zymoresearch.com) 从颗粒中提取 DNA，并通过 Qubit 进行定量。使用 25 ng 尿细胞沉淀 DNA (cpDNA) 和 Nonacus Cell3Target (Nonacus.com) 制备基于捕获的文库。目标富集是通过一个覆盖 c.10 kb 的定制面板，包括 29 个基因的热点/区域：23 个基因的编码区加上TERT启动子和另外五个非编码突变热点，如先前通过大规模肿瘤测序定义的那样。1、4 ]。在 Illumina Nextseq 系统（2 × 150 bp）上对文库进行测序；使用BWA将读数与 hg19 基因组版本对齐. 变异等位基因频率 (VAF) 是使用 bam-readcount 工具提取的，用于先前确定的疾病相关单核苷酸变异 (SNV) [ 1, 4 ]。我们认为 0.5% VAF 是 SNV [ 5 ] 的检测限，而 1% VAF 是定量限。我们报告了每位患者在任何时间超过 1% VAF 的所有 SNV 在治疗期间尿 SNV 的 VAF 的定量变化。平均原始和共识读取深度分别为 27 100 倍和 3000 倍。

使用 AmpliseqHD 试剂 (ThermoFisher) 和工作流程制备基于多重 PCR 的文库。两组目标特异性引物和 2 × 20 ng 尿液 cpDNA 用于扩增与基于捕获的方法相同的目标区域。在 S5 Ion Torrent 系统（2 × 100 bp；ThermoFisher）上使用 540 个芯片对文库进行测序。使用Ion Reporter软件进行比对、共识建立和变异调用。平均原始和共识读取深度分别为 79 300 倍和 5500 倍。我们排除了TERT启动子的 AmpliseqHD 数据，因为这种富含 GC 的扩增子没有为可靠的变异调用提供足够的共识读数。

通过这种方式，在 6 名患者中纵向确定了常见的 UBC 相关突变。对于一名患者，在任何时间点均未检测到突变。对于编码和TERT启动子突变，五分之四的患者的 cpDNA 在基线时（经尿道切除术后、放化疗前）含有高水平的肿瘤 DNA（VAF 4-20%）。到治疗结束时，所有患者的 cpDNA 中的 VAF 均降低至较低水平（图 1）。

在患者 A 中，在基线时检测到TERT启动子的多个突变 > 20% VAF。Sanger 测序证实了 242/243 和 250 TERT突变的异常共现（数据未显示）。这些突变在西妥昔单抗负荷 1 周和放疗 1 周后在较高 VAF 处检测到，然后迅速下降至约 5% VAF，在治疗后期仍可清楚检测到，并且在治疗完成后以 1.4% VAF 存在. 7 个月后，患者 A 局部复发（等级/分期未知）复发。

在患者 B 中，基线时存在TERT 228A 和FGFR3 S249C 突变；两者在治疗后都无法检测到。患者 B 在治疗后 16 个月保持无病。

在患者 C 中，TERT和TP53突变在基线时存在，尽管呈现下降趋势，但在治疗后仍可检测到 1.7% VAF。患者 C 在治疗后 3 个月被诊断为恶性腹水。

在患者 D 中，TERT和TP53突变在基线时存在，在治疗期间迅速下降，并且在治疗完成后检测不到。患者 D 在治疗后 5 个月被诊断为局部复发 (G3pT1)。

在患者 E 中，在基线低 VAF 时存在TERT启动子突变；随着时间的推移，这种突变没有显示出明显的趋势，并且在大多数时间点仍然可以检测到。患者 E 在治疗后 9 个月被诊断为局部复发 (G3pT1)。

基于 PCR 的文库制备 (AmpliseqHD) 与 Ion Torrent 测序相结合，验证了 cpDNA 中基于 Illumina 的突变检测和定量，除了扩增不佳的 TERT 启动子（数据未显示）。两种方法测量的 VAF 相关性良好 ( r ² = 0.96)，AmpliseqHD 分别在 ≥0.5%、1% 和 2% VAF 时证实了通过基于捕获的测序检测到的 84%、94% 和 98% 的 SNV。手动检查所有 cpDNA 的拷贝数配置文件（来自基于捕获的目标富集的脱靶读数的副产品）；拷贝数变异水平反映了 SNV 水平（数据未显示）。

总之，四分之二的复发患者（三名局部，一名远处，完成治疗后 3-9 个月）在治疗完成时无法检测到尿 VAF。鉴于在其他癌症环境中，液体活检样本中的“突变清除”与显着改善的结果有关 [ 6 ] ，因此这一发现对于治疗完成后三分之二的膀胱复发且无法检测到尿 VAF 尤其令人惊讶。推论是治疗完成时可检测到突变的三名患者中有两名在 7 个月内复发。尽管如此，我们建议对放疗后 MIBC 患者进行基于尿液的液体活检监测仍然具有挑战性，应与血浆 ctDNA 监测相结合 [ 7]，或原发性肿瘤组织测序（以允许具有低得多的检测阈值的个性化尿液液体活检 [ 8 ]），或两者兼而有之。在方法学上，基于靶向捕获和基于 PCR 的文库制备和下一代测序均可用于识别尿 cpDNA 中常见的 BC 相关突变。这些初步数据表明需要在这个特定的 MIBC 环境中进行进一步的液体活检研究。