Exome-wide molecular insights from blood and urine liquid biopsies in genitourinary cancers

Abstract

Background: Liquid biopsy has emerged as a non-invasive method for real-time cancer monitoring especially in genitourinary (GU) oncology. Most current studies utilize a panel-based molecular profiling ranging from 50–600 genes; however, a comprehensive exome-wide profiling of real-world patient samples has been lacking.

Methods: Over 2000 liquid biopsy samples were analyzed in this study, including urine samples from early-stage bladder cancer and plasma samples from prostate, lung, breast, esophageal, head and neck cancers, among others. Cell-free DNA (cfDNA) was extracted from these samples and analyzed using PredicineWES+™, a boosted comprehensive whole-exome sequencing (WES) assay with an in-depth coverage of 600 cancer-related genes derived from the PredicineATLAS™ panel. Data analysis was conducted in-house using Predicine's DeepSea bioinformatics software.

Results: The PredicineWES+™ assay demonstrated high sensitivity for detecting somatic mutations across the exome and showed comparable tumor mutational burden (TMB) estimates with the PredicineATLAS™ panel. Interestingly, the highest tumor TMB score was observed in bladder cancer among the analyzed cancers, which is consistent with literature using tissue-based genomic profiling. The most common cancer variants include TP53, ERBB2, KRAS, PIK3CA, FGFR3, APC, among others.

Conclusion: Liquid biopsy-based genomic profiling across various cancer types provides an in-depth analysis of biomarker discovery for personalized cancer care, setting the foundation for improved cancer diagnosis and personalized treatment strategies for urological diseases.

Graphical abstract

Keywords

cell free DNA / genitourinary oncology / liquid biopsy / next-generation sequencing (NGS)

1 INTRODUCTION

Cancer is fundamentally driven by the accumulation of genomic alterations, which can now be monitored through cell-free DNA (cfDNA) analysis using liquid biopsy[1–7]. This approach is particularly promising in genitourinary (GU) oncology, where traditional diagnostic methods often rely on invasive procedures or may be limited by insufficient or inaccessible tissue samples. Tissue biopsy is the prevailing gold standard for molecular profiling and personalized cancer treatment. However, there is an unmet need for a tissue-independent molecular diagnostic solution when tissue is inadequate or unsafe to obtain, especially for urological diseases such as cancers of the bladder, prostate, and kidney.

Liquid biopsies have emerged as a promising non-invasive alternative to tissue biopsies, capturing the genetic landscape of both primary and metastatic tumors[8,9]. The development of liquid biopsy assays, fueled by advances in detecting and characterizing circulating tumor DNA (ctDNA), has led to their gradual introduction in clinical practice; improved ctDNA analysis enables more accurate and less invasive cancer diagnostics, paving the way for clinical trials that integrate these assays[2,5,10–15].

However, current clinical practices using targeted gene panels focus on a limited number of cancer-related genes in tumor tissues and liquid biopsy samples, which can restrict the detection of rarer mutations that may contribute to tumor progression or resistance to therapy. In comparison, whole-exome sequencing (WES) provides a more comprehensive view of the mutational landscape of over 20 000 genes across the exome, capturing both common and rare variants that can significantly impact cancer management and treatment decision-making[16–21].

Tissue-based WES provides comprehensive insights into the coding sequence, allowing for unbiased detection of copy number alterations (CNAs) and precise measurement of complex biomarkers such as tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). WES has been applied across diverse fields, demonstrating its utility in population health, cancer research, and clinical genomic profiling, as detailed in the following paragraphs[16,18,19].

In population health, WES enables large-scale genetic studies that reveal clinically relevant variations. A UK study of nearly 50 000 individuals uncovered four million coding variants, with 98.6% having a frequency below 1%. The study also found that 2% of participants had medically actionable pathogenic variants, such as BRCA mutations. These findings further demonstrate WES's unique ability to identify rare variants with significant health implications, supporting both individual and population-level health strategies[19].

In cancer research, WES offers high-resolution insights into exonic mutations critical for identifying cancer drivers. A joint study by The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) compared WES with whole-genome sequencing (WGS) in 746 cancer patients. While WGS captured more exonic variation and additional mutations in high-GC regions, WES efficiently identified 80% of mutations in exonic regions, balancing coverage and cost-effectiveness. This makes WES a more effective tool for detecting cancer-related mutations in clinical settings[16].

Finally, a comparative study across five institutions highlighted WES's reliability for clinical genomic profiling. The study reported high concordance (91%–95% positive percentage agreement) in somatic variant detection and strong inter-institutional agreement on complex biomarkers like HRD and TMB, despite variability in CNA detection. These results demonstrate WES's robustness for clinical use, provided that standardized quality control measures are in place to ensure consistent, high-quality data[18].

Whole-exome sequencing has become increasingly valuable in cancer research and clinical practice, yet tissue samples, which are the foundation of traditional WES studies, can be difficult to obtain and thus limit real-time analysis. To address this, we used the PredicineWES+™ assay, designed for comprehensive profiling of liquid biopsy samples, including blood and urine, in addition to tumor tissue. This assay provides enhanced coverage of 600 cancer-related genes as well as broad exome coverage of 20 000 genes. The ability to analyze circulating cfDNA from blood or urine offers a significant advantage, enabling real-time, non-invasive detection of tumor variants in cancers such as prostate and bladder cancer. This approach facilitates molecular profiling and minimal residual disease (MRD) detection without the need for tissue samples, representing a precise and flexible alternative to traditional methods.

Previous studies have demonstrated the clinical utility of the PredicineWES+™ assay for the detection of genomic alterations, therapy monitoring, MRD tracking, and drug resistance mechanisms[22–25]. For example, a study by Davis et al. demonstrated that PredicineWES+™ can reliably identify high TMB and genome-wide copy number burden (CNB) in hormone receptor-positive breast cancer, which were associated with poor treatment outcomes. The utility of blood-based circulating tumor DNA (ctDNA) and urine-based circulating tumor DNA (utDNA) for identifying molecular response at the MRD level has shown significant promise in targeted therapies for non-small cell lung cancer (NSCLC) and colorectal cancer (CRC)[22,24,26], as well as in immunotherapy for muscle-invasive bladder cancer (MIBC)[23].

PredicineWES+™ enables tissue-free profiling of ctDNA and utDNA, offering a precise alternative to traditional tissue-informed MRD tracking and radiographic assessments, highlighting its advantages for real-time, non-invasive monitoring over conventional approaches.

This study aims to expand on these findings by using the PredicineWES+™ assay for comprehensive cfDNA profiling in plasma and urine samples from over 2000 patients with various cancer types, including colorectal, lung, bladder, and prostate cancers.

2 MATERIALS AND METHODS

Plasma samples were collected from patients diagnosed with a variety of cancers, including lung, bladder, breast, colorectal, pancreatic, esophageal, prostate, and head and neck cancers. Urine samples were specifically collected from patients with bladder cancer. Plasma sample volumes ranged from 1 mL to 5 mL, and urine samples ranged from 20 mL to 40 mL. Multiple timepoints were collected for some patients as part of clinical follow-up. Table 1 lists the sample type collected for each cancer indication used in this study.

Briefly, cfDNA extracted from patient plasma samples, urine samples and germline DNA extracted from peripheral blood mononuclear cells (PBMC) were processed and subjected to library construction. The resulting DNA libraries were sequenced by PredicineSCORE (i.e., PredicineCNB, its platform name) LP-WGS at 5× coverage or further enriched by hybrid capture for sequencing with PredicineWES+™, a combination assay designed for whole exome sequencing along with boosted sequencing of 600 cancer genes covered by the PredicineATLAS™ targeted panel.

Specifically, the PredicineWES+™ cfDNA NGS assay was utilized for comprehensive profiling of genomic alterations. This assay provides boosted coverage at 20 000× depth for 600 cancer-related genes, including the regions covered by the PredicineATLAS™ panel, with a limit of detection (LoD) of 0.25%. The remaining exome is covered at 2500×, with an LoD of 1%. Sequencing was performed using a minimum input of 5 ng of total cfDNA, extracted from either 1 mL–5 mL of plasma or 20 mL–40 mL of urine.

The assay detects single nucleotide variants (SNVs), small insertions and deletions (indels), gene-level copy number alterations (CNAs), and targeted gene rearrangements or fusions. PredicineWES+™ assay derives TMB scores reporting the total number of somatic mutations detected per megabase of DNA and analyzes mutational signatures and oncogenic signaling pathway involvement. TMB scores were also derived from sequencing data generated by analysis using the targeted 600-gene PredicineATLAS™ to compare bTMB values generated by Predicine™[27]. MSI and tumor fraction were also reported. Sequencing data were processed and analyzed using Predicine's proprietary DeepSEA bioinformatics pipeline, which performs variant calling and calculates additional molecular features using machine learning techniques[25].

Optional concurrent PredicineSCORE low-pass whole-genome sequencing can be performed without additional sample volume to assess blood-based genome-wide copy number burden (bCNB) and tumor fraction, providing further insights into the tumor's molecular characteristics. This assay was designed to capture clinically actionable mutations and provide comprehensive genomic profiling of cancer-related variants across a wide range of solid tumors. PredicineSCORE sequencing data were evaluated to generate bCNB scores representing a comprehensive genome-wide measure of copy number variations (CNVs), including amplifications and deletions across the entire genome adapted from the previously developed ichorCNA method[25].

TMB was calculated as the number of non-synonymous mutations per megabase of sequenced exonic DNA. Higher TMB scores have been linked to increased responsiveness to immune checkpoint inhibitors, particularly in hypermutated cancers such as colorectal and lung cancers. In this study, TMB scores were compared across different cancer types, with bladder and lung cancers exhibiting higher TMB scores relative to esophageal, breast, and head and neck cancers[25].

Bioinformatics and statistical analysis is conducted using R version 4.0.0 and graphic plotting with ggplot2 (RRID:SCR_014601) and ComplexHeatmap(RRID:SCR_017270) packages[28]. The R package pROC was used to perform ROC analysis. The optimal cut-off point was defined on the basis of the greatest Youden index (sensitivity + specificity − 1). The copy number G-score was calculated by the GISTIC (RRID:SCR_000151) 2.0 pipeline via GenePattern (RRID:SCR_003201). The Wilcoxon rank-sum test or Student t-test was used to compare numeric variables. Fisher exact test was used to compare categorical variables. All tests were two sided and considered statistically significant at p < 0.05[23].

3 RESULTS

The PredicineWES+™ assay was applied to over 2000 samples of cfDNA extracted from plasma and urine, and collected from patients with various cancer indications as listed in Table 1. The assay, which provides boosted coverage of 600 cancer-related genes (at ×20 000 depth) and 2500× coverage across the exome, enabled comprehensive whole-exome sequencing for mutation profiling (Figure 1).

Using Predicine's in-house DeepSEA machine learning-based bioinformatics pipeline, the assay successfully identified single nucleotide variants, small insertions and deletions, gene-level copy number alterations, and targeted structural rearrangements. Additionally, key genomic metrics, including microsatellite instability, tumor mutational burden, and tumor fraction, were calculated to provide a more detailed understanding of the tumor's genetic landscape. Figure 1 illustrates the described workflow starting with patient samples and resulting in biomarker reporting for further clinical action.

The PredicineWES+™ assay demonstrated a high sensitivity in detecting mutations across cancer types, including common driver mutations such as KRAS, TP53, and PIK3CA. In plasma-based pan-cancer profiling, common variants were identified, with KRAS detected in 78% of the samples and TP53 in 67% (Figure 2). Of greatest interest for this paper, in urine-based molecular profiling of early-stage bladder cancer, the most frequently mutated genes included TP53 (62%), KMT2D (43%), ARID1A (37%), and ERBB2 (36%) (Figure 3). Interestingly, we observed mutations in CHIP-related genes including DNMT3A, TET2, and ASXL1, which were also observed in the urine-based bladder cancer results; this is likely related to urine hemolysis, often observed in bladder cancer patients. Figures 2 and 3 are oncoplots generated to illustrate the mutational landscape across different cancer types. These plots provide a comprehensive view of the most mutated genes across all patient samples, ranked by incidence, to depict key genomic alterations relevant to precision oncology.

A differential analysis of TMB scores across cancer types using both plasma and urine samples revealed higher TMB scores in bladder and lung cancers compared to other cancers, such as esophageal and breast cancers (Figure 4). This finding aligns with the existing literature on tissue-based TMB measurements. The median TMB values were generally below 10 mutations per megabase across all cancer types, with bladder cancer specifically showing the highest values. These results highlight the potential of cfDNA-derived TMB as a biomarker for identifying patients who may benefit from immune checkpoint inhibitors, particularly in cancers with higher mutational loads.

The high sensitivity and broad coverage of the PredicineWES+™ assay enabled the detection of somatic mutations across the exome, identifying both common and rare mutations that are often missed by smaller gene panels. The results emphasize the utility of this liquid biopsy approach in providing real-time, non-invasive molecular profiling for GU cancer management. The study also demonstrated a strong correlation between blood TMB results obtained with PredicineWES+™ and PredicineATLAS™, supporting the interchangeable use of both assays in assessing TMB for immunotherapy selection. Figure 5 outlines a proposed clinical workflow integrating the liquid biopsy technologies to enable more personalized cancer care.

4 DISCUSSION

To our knowledge, this study represents the first comprehensive liquid biopsy analysis of over 2000 solid tumor samples using boosted whole-exome sequencing, providing real-world evidence for non-invasive personalized cancer care in GU oncology. Consistent with the literature, the identification of liquid biopsy-based cancer variants across a wide range of solid tumors highlights the potential of WES-based cfDNA profiling for extensive genomic analysis for urological disease management[8,9,16,17,25,29].

In an NGS study on tissue-blood TMB concordance in stage III NSCLC patients undergoing concurrent chemoradiotherapy, baseline tissue and blood TMB levels from 15 patients showed a strong positive Pearson correlation of 0.937. As treatment progressed, nearly all mutations were significantly reduced, indicating a clear treatment response. These findings suggest that in patients with locally advanced NSCLC, blood TMB is a reliable surrogate for tissue TMB, and its dynamic monitoring may help identify those who would benefit most from consolidation immunotherapy[9].

TMB in ctDNA has also shown promise as a predictor of benefit from PD-L1/PD-1 inhibitors in retrospective studies[9,29–31]. Consistent with this, the B-F1RST study (NCT02848651), an open-label phase 2 trial, demonstrated that blood TMB is a valuable predictive biomarker for first-line atezolizumab monotherapy in patients with locally advanced or metastatic stage IIIB-IVB non-small cell lung cancer[30].

Additionally, the PredicineWES+™ assay has demonstrated its versatility in detecting variants across the exome, calculating blood TMB[9,25,29–31], and enabling tissue-agnostic, personalized tracking of MRD, offering valuable insights for treatment decisions and tailored cancer care. In a Phase I clinical trial of divarasib for patients with KRAS G12C-positive solid tumors, including lung and colorectal cancers, the assay effectively monitored ctDNA dynamics; patients who responded to treatment showed a decrease in the KRAS G12C variant allele frequency, demonstrating the assay's utility in assessing therapeutic response.

In this study, PredicineWES+™ assay also identified actionable mutations in genes such as KRAS G12C, PIK3CA, ERBB2, and FGFR, underscoring its clinical relevance for targeted therapies. This information enables the identification of patients likely to benefit from FDA-approved therapies, including KRAS G12C-targeting drugs like Adagrasib[32,33] and Sotorasib[34]; PIK3CA-targeted treatments such as Inavolisib[35], Alpelisib[36], and Capivasertib[37]; FGFR-targeted therapy Erdafitibib[38–40], and ERBB2-targeting drugs including trastuzumab (Herceptin), pertuzumab (Perjeta), lapatinib, margetuximab (Margenza), tucatinib, and fam-trastuzumab deruxtecan (Enhertu)[41].

Moreover, the study demonstrates that blood-derived ctDNA and urine-derived utDNA serve as valuable surrogates for tissue-based testing in GU cancers. This observation is supported by studies indicating that liquid biopsies can provide critical information for cancer diagnosis, staging, and monitoring. The non-invasive nature of liquid biopsies is conducive to real-time monitoring of tumor dynamics, which is particularly beneficial for patients where performing tissue biopsies is traditionally challenging or risky.

The high concordance of blood TMB results obtained from PredicineWES+™ and the PredicineATLAS™ panel further support the previous finding that both assays can be used interchangeably for assessing blood TMB. This is particularly important for immunotherapy selection, as blood TMB is increasingly being recognized as a biomarker for responsiveness to immune checkpoint inhibitors. Accurate measurement of TMB through non-invasive liquid biopsies could enhance patient selection for immunotherapy, potentially improving clinical outcomes for patients with GU cancers.

More importantly, the application of liquid biopsy assays such as PredicineWES+ assay in this study represents a significant advancement in improving diagnostic accuracy and personalizing treatment strategies for urological diseases. This comprehensive liquid biopsy platform enables baseline-informed MRD tracking, offering a non-invasive and highly sensitive method to detect residual cancer cells and monitor tumor dynamics. Such capabilities are particularly valuable in muscle-invasive bladder cancer (MIBC), where traditional diagnostic approaches often rely on invasive procedures like cystectomy. By integrating urine-informed MRD tracking, liquid biopsy testing can identify patients who are likely to benefit from bladder-sparing strategies, including immunotherapy, thereby avoiding the need for bladder removal[23]. This is a critical development, as bladder preservation addresses a major unmet need in MIBC, significantly improving patient quality of life and long-term outcomes.

Furthermore, the broader utility of baseline liquid biopsy-informed MRD tracking has been demonstrated in pivotal studies, showcasing its role in predicting recurrence and guiding treatment decisions. Further studies emphasize the non-invasive nature of urine-based approaches, particularly in MIBC where tissue biopsies may be insufficient or infeasible[22,24,26,27]. The high-resolution genomic profiling enabled by PredicineWES+ allows for the identification of actionable biomarkers and facilitates patient stratification for personalized treatment plans. Its precision in tracking MRD enables real-time adjustments to therapeutic strategies, ensuring treatments are both effective and tailored to the molecular profile of each patient.

In the broader context of GU oncology, these technologies have the potential to redefine standard care paradigms by enabling earlier detection of cancer recurrence and optimizing treatment selection, ultimately improving overall survival. The integration of urine-based liquid biopsy solutions into clinical workflows represents a major shift in how we approach the diagnosis and management of complex urological cancers in the near future.

Despite the promising results, this study has limitations. The use of random real-world samples introduces variability in patient selection and sample quality, which may affect the generalizability of the findings. Additionally, the lack of longitudinal ctDNA follow-up data and the absence of matched tumor tissue samples limit the ability to directly compare liquid biopsy results with tissue-based genomic profiles. Future studies should incorporate longitudinal monitoring and include matched tissue samples to validate and expand upon these findings.

5 CONCLUSIONS

The current molecular profiling study of 2000 blood and urine samples represents a significant advancement in precision medicine, providing the ultimate comprehensive molecular ctDNA profiling across the whole exome with boosted coverage of 600 cancer-related genes. This study highlights the utility of the various types of liquid biopsy assays for detecting clinically actionable mutations, calculating TMB, and enabling tissue-agnostic personalized MRD tracking, making it a valuable tool for personalized cancer management and clinical drug development. The testing of plasma and urinary cfDNA analysis provides a comprehensive view of the patient's mutational landscape and offers a feasible option for personalized MRD tracking for cancer patients. By enabling real-time assessment of treatment response and disease progression, blood- and urine-based liquid biopsy have the potential to significantly enhance the personalization of cancer care and precision medicine, especially in GU oncology.

More importantly, the FDA's recent guidance on the use of ctDNA biomarkers for drug development in curative-intent solid tumors lends serious credence to the potential of liquid biopsy technologies to transform patient care in the near future, as shown in Figure 5. This pivotal endorsement reflects the increasing recognition of MRD tracking as a critical biomarker for early detection, monitoring disease progression, and guiding more personalized treatment strategies. Our study's findings align directly with the FDA's vision by demonstrating the clinical utility of blood- and urine-based liquid biopsies in GU oncology, fulfilling urgent unmet needs in the clinical domain. The demonstrated ability of urine-based MRD tracking to identify bladder cancer patients who, for example, may benefit from bladder-sparing approaches, such as immunotherapies, without invasive cystectomy exemplifies the assay's potential to revolutionize care and improve both overall survival and quality of life for bladder cancer patients. These advancements are paving the way for non-invasive, real-time biomarkers to refine clinical treatment decisions and improve patient outcomes.

By integrating these technologies into clinical workflows, oncology care is positioned for a fundamental change, particularly in dealing with complex cancers such as those in GU oncology. The introduction of both blood- and urine-based liquid biopsy solutions enables a significantly more convenient and non-invasive approach to diagnosis and management, ultimately improving outcomes and advancing the promise of precision medicine. This study has demonstrated not only the technological advancements but also the serious clinical implications of molecular diagnostics for advancing personalized care in GU oncology.

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