The Current Role of Circulating Biomarkers in Non-Muscle Invasive Bladder Cancer

Michael Rink; Heidi Schwarzenbach; Malte W. Vetterlein; Sabine Riethdorf; Armin Soave; on behalf of the EAU Young Academic Urologists—Urothelial Cancer Working party


Transl Androl Urol. 2019;8(1):61-75. 

In This Article


Numerous urinary tests (e.g., BTA stat, UroVysion (FISH), Immunocyt, NMP22, etc.) have been developed in the past, driven by the low sensitivity of urine cytology.[2] Indeed, there is an urgent need for better prediction of individualized outcomes of NMIBC, particularly of disease recurrence and progression, but also survival endpoints. Currently there are no approved circulating molecular biomarkers for use in the clinic to manage UCB, although several above mentioned commercially available urinary biomarkers have been FDA-approved for NMIBC detection and surveillance.[84] While some of these mainly protein- and DNA-based assays revealed a better sensitivity compared to urine cytology, they largely suffer from insufficiently low specificity. In consequence, they are not routinely recommended by national or European guidelines and usually represent costly patient direct payer services. Still, it is reasonable using urine as source for liquid biomarker analysis as tumor cells may early be released to this source during UCB development and at time of local recurrence.[85] Liquid biopsies represent a non-invasive technique detecting prognostic and predictive circulating biomarker offering information about molecular and phenotypic cancer characteristics. The term "liquid biopsy" is commonly used for analysis of blood-based biomarkers including CTC and ctDNA, which are released into the peripheral blood from the primary tumor and/or metastatic deposits.[85,86] However, recently investigators extended the definition of liquid biopsies also to the urine of NMIBC patients, as they developed personalized assays for disease surveillance based on individual tumor-specific genomic variants.[10] In fact, urine represents the most non-invasive assessable source for biomarker analyses.

Epitopes and genetic material from UCB can frequently be detected in the urine.[87] Nevertheless, it is reasonable investigating and combining information of circulating biomarker from the urine and the blood, as genomic characteristics may vary between the primary tumor and distant sites[88] and, in addition, may represent different clones with variable potential for disease progression and metastasis. For these investigations, different circulating biomarker sources may be used: While CTC and ctDNA can be detected and analyzed in the peripheral blood circulation, in the urine cell-free tumor DNA represents the ideal substrate. Different studies demonstrated that urinary DNA in UCB patients was highly characteristic for DNA derived from tumors.[84] The clinical relevance of ctDNA in UCB is sustained by its high somatic mutation rate, whose detection may be informative for disease surveillance at different stages and different times. cfDNA from urine had a higher tumor genome border and allowed greater detection (90%) of key somatic mutations in than cellular DNA from urine.[84,89] In addition, the size of cfDNA may indicate its source of origin. Apoptotic cells produce DNA fragments of 180–200 bp or multiples of this unit, whereas necrotic cells release higher molecular-weight DNA fragments in size of over 10,000 bp.[14] Considering its quality, the different cfDNA fragment lengths have important implications in the measurement and analysis of ctDNA. For example, as reported by Ellinger et al., the fragments of mitochondrial DNA (mtDNA) are somewhat longer in UCB patients. These researchers found that the integrity defined as ratio of mtDNA-220 to mtDNA-79 fragments was increased in serum of UCB patients compared to control subjects and prostate cancer patients.[90] Indeed, investigations of ctDNA in the blood stream as supplement or alternative to urine analyses are of particular relevance, as the genomic pool and subsequently the tumorbiologic potential may vary between both sources. Estimations indicate that a significant amount of up to 3.3% of tumor DNA is released into the bloodstream every day depending on the tumors size.[26] The amount of ctDNA in the whole pool of cfDNA containing both tumor and normal cfDNA may significantly vary from 0.01% to 50% among cancer patients, and be related to tumor size. In MIBC patients, the ctDNA fraction may even increase above 50% of cfDNA. In contrast, average cfDNA yields in MIBC before therapy seem to be less than 10 ng per mL of plasma (representing only 1,500 diploid genomes).[91] Thus, the levels of cfDNA that correlate with changes in tumor burden have a great dynamic range, even greater than CTC. In human blood, this cfDNA circulates predominantly as nucleosomes[92] whose histone modifications may also be tumor-specific.[93] From blood, cfDNA is removed by the liver and kidney, and its half-life is variable ranging from 15 minutes to several hours.[26]

CTC are usually assumed being surrogates for micrometastatic disease or minimal residual disease (MRD). The risk for development of lymph node or distant metastasis is clearly correlated to the tumor stage in UCB.[94] Indeed, the prognostic value of CTC has been demonstrated in MIBC and advanced UCB.[12,24,95] In muscle-invasive and advanced bladder cancer CTC are associated with inferior cancer-specific and overall survival. Intuitively it feels reasonable that the presence of CTC as micrometastatic disease is associated with these two important cancer-related outcomes. Despite overall and cancer-specific survival are also relevant in NMIBC, the more important outcome endpoints in these early disease stages are rather disease recurrence and disease progression. Still, CTC represent an interesting circulating biomarker also in NMIBC for individually tailored cancer outcome prognostication as well as potentially tumor staging.[23] Interestingly, even in early NMIBC CTC are found in a significant number of patients. Of great importance, CTC are associated with disease recurrence and progression in a couple of studies.[80–82] Although the biological mechanism between CTC in the peripheral circulation and local tumor recurrence in the bladder are inconclusively understood today, CTC may contain cell clones of the most aggressive tumor parts. These tumor fractions may not only have the potential for systemic spread, but also harbor the potential for local recurrence and progression. In consequence, CTC measurement may help selecting patients at highest risk for an inferior course of disease and thus who may benefit most from early cystectomy or perioperative chemotherapy. However, the scarceness and potentially heterogeneous molecular nature of CTC requires high-throughput capture/enrichment, detection and characterization technologies.[21] In general, in non-metastatic UCB, and particularly NMIBC, the number of detected CTC is on average a single cell. Epithelial-mesenchymal transition (EMT) is frequently observed during the metastatic cascade in UCB and is accompanied with a loss of EpCAM and/or CK expression.[20,25] In consequence, common CTC enrichment and enumeration platforms including CellSearch® and CELLection® may miss CTC after EMT, which might be more common in aggressive tumors. In fact, the very low CTC detection rate significantly compromises the possibilities of in-depth genome analyses. Despite the introduction of several new platforms, including flow cytometry-based assays and lab-on-a-chip micro fluidic devices, in the last decade that tended addressing these limitations,[21,70] the low sensitivity of all currently available systems remains a serious clinical limitation.

It is crucial to emphasize further important clinical limitations among studies on liquid biopsies in NMIBC. Most studies are of retrospective nature or it is not entirely clear, whether they were conducted pro- or retrospectively.[71,78,82] In addition, in general the number of included patients is relatively low and follow-up is rather short. In consequence, the number of events is also low, which may have biased results. Of particular importance regarding CTC studies, most studies only focused on high risk NMIBC subtypes, especially pT1 G3 disease.[71,80,82] Indeed, these results do not indiscriminately reflect the entire biological range of NMIBC and therefore these findings cannot be extrapolated to other NMIBC including pTa, G1-G3 and CIS, respectively. CTC studies with serial measurements following TURBT and/or during intravesical instillation therapy are warranted.