This is an open access article under the terms of a license that permits non-commercial use, provided the original work is properly cited. © 2022 The Authors. Société Internationale d'Urologie Journal, published by the Société Internationale d'Urologie, Canada. Key Words Competing Interests Article Information Bladder cancer, predictive biomarker, urine biomarker, tissue biomarker, circulating tumor DNA, molecular subtype, immunohistochemistry None declared. Received on September 28, 2021 Accepted on December 4, 2021 This article has been peer reviewed. Soc Int Urol J. 2022;3(4):245–257 DOI: 10.48083/RVZV1144 245SIUJ.ORG SIUJ • Volume 3, Number 4 • July 2022 REVIEW Predictive Biomarkers in the Management of Bladder Cancer: Perspectives in an Evolving Therapeutic Landscape Patrick J. Hensley,1 Niyati Lobo,1 Kelly K. Bree,1 Wei Shen Tan,2 Paolo Gontero,3 Stephen B. Williams,4 Charles C. Guo,5 Gianluca Giannarini,6 Lars Dyrskjøt,7 Ashish M. Kamat1 1 Department of Urology, University of Texas MD Anderson Cancer Center, Houston, United States 2 Department of Urology, London North West University Healthcare NHS Trust, London, United Kingdom 3 Department of Urology, Città della Salute e della Scienza, Molinette University Hospital, Turin, Italy 4 Division of Urology, The University of Texas Medical Branch at Galveston, Galveston, United States 5 Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, United States 6 Urology Unit, Santa Maria della Misericordia University Hospital, Udine, Italy 7 Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark Abstract Bladder cancer (BC) is a heterogeneous disease with prognosis and therapeutic strategies highly dependent on tumor grade and stage. Predictive biomarkers of therapeutic response have been studied to guide selection of intravesical and/or systemic therapy. A predictive biomarker is measured before the start of treatment and provides information on the likelihood of response to a specific therapy.  Many candidate predictive biomarkers for BC have been identified, but few have been rigorously validated or distinguished from simply having treatment-agnostic prognostic capacity. Identifying predictive biomarkers tailored to therapeutic mechanism of action has considerable implications for the sequencing of therapies, as well as bladder preservation strategies in advanced disease states. We evaluate predictive tissue-based, urine-based, and serum-based biomarkers across the spectrum of non–muscle-invasive and muscle-invasive BC and preview predictive biomarkers for emerging targeted therapies. Introduction Biomarker development has undergone an evolution over the years, with increasing focus on predictive biomarkers in the field of bladder cancer. A predictive biomarker is measured before the start of treatment and provides information on the likelihood of response to a specific therapy. While many candidate predictive biomarkers for bladder cancer (BC) therapeutic response have been proposed, few have had their predictive value compared with non-treated cohorts to distinguish them from simply having prognostic capacity. Predictive biomarkers have utility throughout the spectrum of disease in BC, from aiding diagnosis to guiding initial therapy selection, and even to prompting timely abandonment of ineffective treatment in lieu of definitive surgical management, radiotherapy, or other systemic therapy. Our understanding of specific therapeutic mecha- nisms of action is key to designing predictive markers that offer insight into innate tumor biology and therapeutic susceptibility. Measuring the predicted or elicited response to local or systemic therapies provides an opportunity to tailor biomarker development to specific therapies. In this narrative review, we discuss predictive BC tissue-based, urine-based, and serum-based markers (Figure 1), identify current limitations and unmet needs, and define the evolution of biomarker development in the landscape of targeted therapies. We summarize the current state of predictive biomarkers for both NMIBC and MIBC using a non-systematic review of published literature and provide expert opinion on the accuracy and clinical applicability of emerging biomarkers. Predictive Tissue Biomarkers Non–muscle-invasive bladder cancer The most extensively studied tissue-based molecular ma rkers pred ic t ive of t herapeut ic response to intravesical therapy for non–muscle-invasive BC (NMIBC) to date are p53 and Ki67, both potent cell cycle regulators. Dysregulation of the tumor suppressor p53 has been correlated with BC progression, but not recurrence, following BCG therapy[1–3]. However, a prospective study failed to validate p53 as a predictive biomarker[4]. Ki67, a nuclear protein indicative of cell proliferation, has predictive ability for intravesical therapies. The expression of Ki67 has been correlated with recurrence following BCG[5] and both recurrence and progression following intravesical chemotherapy[6]. Table 1 summarizes other molecular biomarkers demonstrated to be predictive of BCG response. These include cell cycle regulators, apoptosis inhibitors, cell adhesion molecules, and proliferative markers[7]. The majority of these biomarkers have been evaluated only retrospectively in small single-center cohorts with non-standardized methods of measurement and without external validation, thus limiting our ability to derive definitive conclusions about their utility as predictive biomarkers. Predictive biomarker panels have also been recently evaluated following intravesical BCG. A subgroup anal- ysis of 2 large Nordic randomized trials performed by Malmström et al. analyzing expression of ezrin, CK20, and Ki67 failed to show a correlation between biomarker expression and recurrence or progression following BCG therapy[8]. Similarly, Park et al. eval- uated the altered expression of 7 potential biomarkers (p53, pRb, PTEN, Ki67, p27, FGFR3, and CD9) and found no predictive value for recurrence or progression among high-grade T1 tumors treated with BCG[9]. These conflicting data underscore the need for compre- hensive validation studies. Functional mutations in DNA mismatch repair genes have also been implicated in predicting therapeutic response in BC. Sanguedolce et al. demonstrated that MutL homologue 1(MLH1) was an independent predic- tor of progression-free survival among patients treated with adequate BCG[10]. In patients who received BCG, polymorphisms within DNA repair pathways have been associated with recurrence-free survival (RFS)[11]. Furthermore, tumor mutational burden has recently been shown to correlate with response to intravesi- cal therapy[12]. These data were corroborated through a comprehensive gene analysis on index non–muscle- invasive tumors, with increased mutational burden noted among high-grade NMIBC[13]. Specifically, ARID1A mutations were predictive of shorter RFS in patients treated with BCG; however, no association was noted between RFS and other analyzed markers, includ- ing TP53, MDM2, ERBB2, and FGFR3 following BCG therapy[13]. Damrauer et al. recently performed RNA-based profiling to identify a novel expression signature of an inflamed tumor microenvironment (TME) which was predictive of BCG response[14]. Notably, molecular subtyping and immune checkpoint gene expression were not predictive of treatment response. However, a pre-treatment TME enriched with CD25+ regulatory T cells and tumor-associated macrophages and decreased Abbreviations BC bladder cancer BCG Bacillus Calmette-Guérin ctDNA circulating tumor DNA NAC neoadjuvant chemotherapy NMIBC non–muscle-invasive bladder cancer MIBC muscle-invasive bladder cancer RFS recurrence-free survival TAM tumor-associated macrophage TMB tumor mutational burden TME tissue microenvironment TURBT transurethral resection of bladder tumor FIGURE 1. Biomarker source material and analysis for NMIBC and MIBC 246 SIUJ • Volume 3, Number 4 • July 2022 SIUJ.ORG REVIEW continued on page 248 247SIUJ.ORG SIUJ • Volume 3, Number 4 • July 2022 Predictive Biomarkers in the Management of Bladder Cancer: Perspectives in an Evolving Therapeutic Landscape TABLE 1. Predictive urine biomarkers for BCG therapeutic response in patients with NMIBC Reference Year Marker Study Populationa Number of patients Detection Method(s) Results Palou et al.[5] 2009 Ezrin, Ki67 HG T1 92 IHC Low ezrin expression (< 20%) associated with increased progression among patients receiving induction BCG (P = 0.031) b Differential expression of Ki67 in patients with early recurrence following induction BCG (P = 0.015) b Zachos et al.[83] 2009 Telomerase reverse transcriptase (hTERT) HG T1 NMIBC 30 IHC hTERT nucleolar staining in >75% of cells was associated with worse RFS following induction BCG (relative risk of recurrence 8.85 [95% CI 1.9–41.6])b Cormio et al. [84] 2010 pRb HG T1 27 IHC Altered pRb expression was predictor of recurrence (P = 0.037) and progression (P = 0.018) in patients treated with adequate BCGc Alvarez-Mugica et al.[85] 2010 Myopodin methylation HG T1 NMIBC 170 Methylation analysis Among patients treated with adequate BCG, myopodin methylation associated with increased recurrence rate (P = 0.011) and progression (P = 0.030)b Shirotake et al.[86] 2011 Angiotensin II Type 1 Receptor (AT1R) NMIBC 79 IHC Strong AT1R expression associated with worse 1-year RFS following BCG(P = 0.0012)b Lima et al.[87] 2013 sialyl-Tn (sTn), sialyl-6-T(s6T) High-risk NMIBC 94 IHC High sTn and s6T expression was associated with BCG response (P = 0.024 and P < 0.0001) and with increased RFS (P = 0.001)c Sen et al.[88] 2014 Nestin HG T1 63 IHC Recurrence rates were higher in nestin(+) compared to nestin(-) among patients receiving induction BCG (60.6% versus 30%, P = 0.014)c Cheng et al.[89] 2015 E2F4 NMIBC 188 RNA sequencing Treatment with BCG in E2F4 score > 0 patients associated with improved progression-free survival (P = 0.06) Raspollini et al.[90] 2016 P16, galectin-3, CD44, CD138, E-cadherin, survivin, HYAL-1, topoisomerase-liα HG T1 (≤ 3cm) 92 IHC TOPO-2α predicted DFS (P = 0.029), surviving predicted PFS (P = 0.020), surviving and E-cad predicted OS (P = 0.006, 0.030)b aAll studies listed were analyses of retrospective cohorts. bMultivariate analysis. cUnivariate analysis. density of Th2-predominant CD4+ T cells was predic- tive of poor RFS following BCG therapy[15]. Lim et al. also noted that TME-tissues from BCG-responders was enriched with active CD8+PD-1(-) T cells and non-reg- ulatory CD4+FOXP3(-) T cells, whereas the TMEs of non-responders were characterized by increased levels of exhausted CD8+PD-1(+) T cells[16]. Among patients with carcinoma in situ (CIS) treated with induction BCG, lower tumor-associated macro- phage (TAM) density was associated with improved recurrence-free survival compared with those with higher TAM density[17]. Furthermore, when subsets of TAMs (M1 and M2) were analyzed, a low density of M1-TAM and high density of M2-TAM were predic- tors of worse disease-specific survival among patients treated with BCG[18]. Other components of the complex immune response to BCG therapy that have been predic- tive of favorable treatment response include increased expression of major histocompatibility complex 1[19], low level of infiltration by tumor-infiltrating dendritic cells[20], and increased levels of natural killer cell recep- tor ligands[21]. Taken together, these data indicate that the TME likely plays an important role in modulating the BCG therapeutic response and serves as a promising predictive biomarker target. While there is a paucity of investigation into predic- tive biomarkers for individual intravesical chemother- apeutic agents in NMIBC, single-institution series suggest high FOXM1 expression[22] and tumors with high proliferation index (as measured by Ki67) achieve favorable responses to mitomycin C[6,23]. Muscle-invasive bladder cancer Cisplatin-based neoadjuvant chemotherapy (NAC) before radical cystectomy confers an overall survival benef it for pat ients w it h M IBC[24]. Genom ic interrogation revealed that a significant proportion of MIBCs harbor mutations in DNA damage repair (DDR) genes. DDR pathways play a critical role in the cellular 248 SIUJ • Volume 3, Number 4 • July 2022 SIUJ.ORG REVIEW , Cont’d TABLE 1. Predictive urine biomarkers for BCG therapeutic response in patients with NMIBC Reference Year Marker Study Populationa Number of patients Detection Method(s) Results Meeks et al.[12] 2016 Cancer- associated gene panel High-risk NMIBC 25 DNA sequencing Increased total mutational burden associated with IO response between non-progressors, progressors and metastatic tumors (15, 10.1, 5.1 mutations/MB, respectively; P = 0.02)c Pietzak et al.[13] 2017 ARID1A NMIBC 65 DNA sequencing ARID1A mutations associated with shorter RFS after BCG (HR 3.14, P = 0.002)b Sanguedolce et al.[10] 2018 MLH1 HG T1 67 IHC MLH1 expression was an independent predictor of PFS among patients treated with adequate BCGb Mano et al.[91] 2018 HSP 60 HSP 70 HSP 90 HG T1 54 IHC HSP70 associated with lower risk of recurrence (HR 0.29, P = 0.003) and progression (HR 0.33, P = 0.045), HSP 60 associated with higher risk of progression (HR 3.96, P = 0.012) among patients treated with at least induction BCGb Shao et al.[92] 2021 Next generation sequencing Intermediate or high-risk NMIBC 58 DNA sequencing NEB, FGFR1, and SDHC were independent predictors of recurrence following BCG (P = 0.001, P = 0.004, and P = 0.017, respectively)b aAll studies listed were analyses of retrospective cohorts. bMultivariate analysis. cUnivariate analysis. response to platinum-based chemotherapy, providing a rationale for their use as predictive biomarkers. ERCC2 encodes a DNA helicase that plays a central role in the nucleotide excision repair pathway, repairing DNA cross-linking caused by genotoxic agents such as platinum chemotherapies. Van Allen et al. performed whole-exome sequencing on pre-treatment tumor and germline DNA from 50 patients with MIBC receiving cisplatin-based NAC before cystectomy[25]. ERCC2 was the only significantly mutated gene enriched in cisplatin-responders compared with non-responders. This finding was mechanistically confirmed in vitro, as expression of wild-type ERCC2 in an ERCC2-deficient BC cell line rescued cisplatin sensitivity. Using an inde- pendent MIBC cohort, Liu et al. found ERCC2 alter- ations in 8/20 responders to chemotherapy (40%) versus 2/28 non-responders (7%) (P = 0.010, OR 8.3 [95% CI 1.4 to 91.4])[26]. In a subsequent phase II trial of neoadju- vant dose-dense gemcitabine and cisplatin in patients with MIBC, the presence of one or more alterations in a panel of 29 DDR genes, including ERCC2, was associ- ated with chemosensitivity (positive predictive value for 15 muta- tions/MB). In contrast to the PURE-01 study and trials in the metastatic setting, the neoadjuvant ABACUS study of the PD-L1 inhibitor atezolizumab was unable to show a significant association between PD-L1 expression (either on tumor cells or infiltrating cells) and therapeu- tic response[48]. The lack of standardization of PD-L1 assessment, such as different antibodies, thresholds for PD-L1 positivity, and immune cell quantification, is likely to contribute to its limited predictive ability in BC. Bandini et al. recently constructed a probability calcula- tor incorporating 2 biomarkers (PD-L1 expression and TMB) and baseline clinical T stage to predict patho- logic complete response after pembrolizumab[49]. This predictive model performed well with a concordance index of 0.77 (95% CI 0.68 to 0.86), highlighting the complexity of the tumor-immune interaction and util- ity of predictive biomarker panels compared with single markers alone. TABLE 2. Clinical trials investigating tissue-based biomarkers predictive of neoadjuvant therapy response in MIBC NCT Trial Name Study Population Biomarker(s) Under Investigation Intervention Primary Endpoints 02710734 Risk Enabled Therapy After Initiating Neoadjuvant Chemotherapy for Bladder Cancer (RETAIN) cT2-3 N0 bladder cancer Sequenced pre-NAC TURBT specimens for DDR mutations TUR followed by accelerated MVACa; patients with complete clinical response and DDR mutation managed with bladder sparing, others treated with intravesical chemotherapy, radiation therapy or radical cystectomy Metastasis-free survival (MFS) at 2 years 03609216 Alliance A031701 cT2-3 N0 bladder cancer Sequenced pre-NAC TURBT specimens for DDR mutations TUR followed by gemcitabine/ cisplatin; patients with DDR mutation and pathologic response (≤ycT1) managed with bladder sparing; others treated with chemoradiation or radical cystectomy Event-free survival (MFS) at 3 years 03558087 HCRN GU 16-257 cT2-4 N0 bladder cancer Sequenced pre-NAC TURBT specimens for DDR mutations and tumor mutational burden TUR followed by gemcitabine/ cisplatin/nivolumab; patients with complete clinical response managed with bladder sparing and maintenance novilumab, others treated with radical cystectomy (1) complete clinical response rate (2) ability of complete clinical response to predict 2-year metastasis-free survival 02177695 SWOG 1314 cT2-4 N0 bladder cancer Co-expression extrapolation (COXEN) gene expression algorithm TUR followed by neoadjuvant dose- dose MVACa or gemcitabine/cisplatin prior to radical cystectomy Assess whether COXEN profile is (1) prognostic of pT0 rate or ≤pT1 at cystectomy and (2) a predictive factor between chemotherapy regimens amethotrexate, vinblastine, doxorubicin, cisplatin 250 SIUJ • Volume 3, Number 4 • July 2022 SIUJ.ORG REVIEW Predictive Urine Biomarkers Non–muscle-invasive bladder cancer Urine is a uniquely qualified biomarker source material, as it is readily available, easily collected, and has direct tumor contact. Urine-based biomarkers have been primarily studied for purposes of diagnosis and surveillance of BC, with relatively few having sufficient accuracy to predict therapeutic response. The true predictive biomarker capacity of the urine-based markers mentioned herein remain largely uncharacterized as many were studied exclusively in treated populations or measured as an elicited response after intravesical therapy. BCG has proven efficacy in reducing recurrence and progression in intermediate and high-risk non– muscle-invasive BC (NMIBC)[50]. Reliable biomark- ers predictive of BCG therapeutic response could have tremendous implications in sequencing of therapy for NMIBC. Unfortunately, given the relative non-specific mechanism of action and elicited immune response by BCG, clinicopathologic factors such as tumor stage, grade, size, presence or absence of CIS, tumor focality and recurrence history remain the most reliable predic- tors of BCG therapeutic response[51]. The rationale for several candidate preclinical biomarkers have employed the mechanism of BCG therapeutic response[52]. Interleukin (IL)-8 is one of the first cytokines with induced expression after BCG therapy. In a pilot study of 20 patients, high levels of IL-8 expression measured 6 hours after BCG instillation had lower rates of recurrence and progression[53]. Addition- ally, failure to induce expression of IL-2 and IL-18 after BCG has been associated with poor BCG therapeutic response[54]. Because BCG immunogenicity is complex and non- specific, single candidate markers alone may be unreli- able predictive tools. The Cytokine Panel for Response to Intravesical Therapy (CyPRIT) nomogram was gener- ated from expression profiling of 9 inducible urinary cytokines (IL-2, IL-6, IL-8, IL-18, IL-1ra, TRAIL, IFN-γ, IL-12[p70], and TNF-α) in 130 patients with NMIBC at the MD Anderson Cancer Center using an enzyme- linked immunosorbent assay (ELISA) at baseline and at specified time points throughout BCG therapy[55]. This nomogram predicted the likelihood of recurrence with 85.5% accuracy. Additionally, baseline levels of pro-tumorigenic cytokines were profiled pre-treatment. Indeed, expression of IL-8 in urine was associated with recurrence in BCG-treated patients, with patients who had higher baseline urinary IL-8 levels experiencing a 4-fold increased risk of tumor recurrence[56]. Inter- estingly, higher baseline levels of IL-8 expression in peripheral blood leukocytes similarly correlated with disease recurrence, suggesting a role for this cytokine as a systemic marker for BCG immunogenicity and thera- peutic response. Contrasting these results to the afore- mentioned studies indicating that induced urinary IL-8 expression after BCG instillation is a marker of disease recurrence and progression, these data highlight the complexity of baseline and elicited immune states, as well as the stability and kinetics of cytokine profiling, in determining their potential as predictive biomarkers. The Oncuria urine-based assay measures the expres- sion of cancer-associated markers in voided urine speci- mens[57]. Using urine samples from the CyPRIT cohort, investigators found that pre-treatment concentrations of MMP9, VEGFA, CA9, SDC1, PAI1, APOE, A1AT, ANG, and MMP10 were increased in subjects with disease recurrence. A predictive model of treatment outcomes reached an area under the receiver operating curve of 0.89 (95% CI 0.80 to 0.99), with a test sensitivity of 81.8% and a specificity of 84.9%. While not specifically FDA approved for this indica- tion, the fluorescence in situ hybridisation (FISH) assay, which detects aneuploidy in chromosomes 3, 7, and 17 and loss of the 9p21 locus in voided urine samples (UroVysion), has been studied in the context of BCG therapeutic response. In a study of 37 patients primar- ily receiving BCG for NMIBC, 100% of patients with a positive post-treatment UroVysion FISH developed tumor recurrence[58]. The predictive capacity of positive post-treatment UroVysion was independently confirmed in several studies with variable adjuvant intravesical agents for NMIBC[59–62]. A subset of “molecular BCG failure” patients based on mid-treatment persistence of a positive FISH assay was subsequently defined. In a study of 126 patients, those with a positive FISH test during therapy were 3 to 5 times more likely to develop recurrence and 5 to 13 times more likely to progress compared with patients with negative mid-treatment FISH[63]. This was subse- quently validated in an independent, multicenter trial where FISH was predictive of recurrence and/or progres- sion events at baseline (HR 2.59; 95% CI 1.42 to 4.73), before the sixth induction instillation (HR 1.94; 95% CI 1.04 to 3.59) and at 3-month follow-up (HR 3.22; 95% CI 1.65 to 6.27)[64]. Defined as positive FISH at 6 weeks and 3 months after induction BCG in the setting of a negative cystoscopic evaluation, this molecular failure denotes a group at high risk of stage progression if managed with further BCG therapy, and who should be considered for enrolment into clinical trials or timely cystectomy[65]. Muscle-invasive bladder cancer There currently exist no urine-based biomarkers to reliably predict therapeutic response in MIBC. However, broad genomic expression and mutational profiling of molecular targets of novel therapeutic agents, including 251SIUJ.ORG SIUJ • Volume 3, Number 4 • July 2022 Predictive Biomarkers in the Management of Bladder Cancer: Perspectives in an Evolving Therapeutic Landscape monoclonal antibodies and antibody-drug conjugates, have emerging rationale. For example, UroSEEK is a urine-based molecular assay which detects alterations in 11 commonly mutated genes which are druggable targets: TERT, FGFR3, PIK3CA, TP53, HRAS, KRAS, ERBB2, CDKN2A, MET, MLL, and VHL[66]. As sequencing technology becomes more refined, urine-based genetic material, including exfoliated tumor cells, cell-free DNA, and exosomes may prove feasible sources for molecular subtyping and further predictive biomarker development for MIBC. Predictive Serum Biomarkers Serum biomarkers for BC remain an area of active research. These liquid biopsy tests may have a role in cancer risk stratification, characterization of tumor molecular signatures, and predicting response to systemic treatment, as well as for cancer surveillance. To date, these tests have remained proof-of-concept in preclinical studies but have emerging clinical relevance to guide treatment decisions. Circulating tumor cells (CTC) represent one of the first studied serum biomarkers. While they have a poor sensitivity of 35% for the detection of BC due to their scarcity in circulating blood, the presence of CTCs has been associated with higher histological stage, grade, lymph node involvement, and presence of metastatic disease[67,68]. In the pre-radical cystec- tomy setting, CTCs have been shown to predict poor oncological outcomes, independent of clinicopatho- logical variables[69]. Serum RNA markers such as long non-coding RNAs (IncRNAs) and microRNAs (miRNA) have been reported to have prognostic value. Zhang et al. reported that high serum UBC1 expression was associated with lower NMIBC RFS (P = 0.01) [70]. In a systematic review and meta-analysis of 26 studies, 6 miRNA (miR-21, miR-143, miR-155, miR-214, and miR-222) were identi- fied as being predictive of early disease recurrence and progression[71]. More recently, there have been rapid advancements in circulating tumor DNA (ctDNA). Developments in deep-sequencing technology have allowed for the reli- able identification of double strand DNA fragments as small as 150 bp. Birkenkamp-Demtröder et al. devel- oped personalized ctDNA assays based on NGS of tumor tissue. They report that ctDNA was present even in NMIBC patients, and the presence of higher levels of ctDNA was associated with subsequent disease progres- sion and metastasis[72]. In patients undergoing radical cystectomy, ctDNA predicted oncological outcomes in several settings[31]. Patients positive for ctDNA at diag- nosis before NAC had a higher 12-month recurrence rate (42% versus 0%)[31]. Similarly, following NAC, patients positive for ctDNA had a higher rate of 12-month disease recurrence (75% versus 7%) than did ctDNA negative patients[31]. Additionally, in the surveillance setting, ctDNA had a median lead time of 96 days over radio- logical imaging[31]. This lead time of ctDNA detection before radiologic or symptomatic clinical detection is allowing investigators to define a “biochemical relapse” to guide timely initiation of first-line atezolizumab after RC in a clinical trial setting (NCT04138628). The role of ctDNA as a predictive biomarker for atezolizumab has also been reported in a study where patients with ctDNA positivity had a significantly improved overall survival compared with the observational arm (HR: 0.59; 95% CI 0.41 to 0.79)[73]. Unmet Needs in Biomarker Development Characteristics for the ideal biomarker predictive of therapeutic response vary considerably by disease stage. For NMIBC, BCG is the gold standard intravesi- cal treatment because of its efficacy, favorable cost, and tolerability. To date, biomarkers predictive of response to BCG have primarily focused on identifying early non-responders in an effort to transition them to off-la- bel salvage intravesical chemotherapy options or timely radical cystectomy. However, in the era of BCG short- ages and emerging intravesical and systemic therapies available in earlier disease states, we would benefit from predictive markers that could guide initial therapeutic response. With emphasis on bladder preserving strate- gies, it will become equally important to identify predic- tive biomarkers of salvage intravesical and systemic therapies after BCG failure. Recent molecular classification of NMIBC has correlated candidate molecular subtypes to innate sensi- tivity and resistance to BCG therapy, and provided ther- apeutic rationale for upfront use of FGFR inhibitors, ICIs, or intravesical chemotherapy[74]. Lastly, favorable results have recently been reported in the Phase III trial of intravesical nadofaragene firadenovec (rAd-IFNa/ Syn3) for BCG unresponsive NMIBC[75]. These investi- gators are validating a serum-based adenoviral antibody titer assay to evaluate immunogenicity of the gene ther- apy and corresponding therapeutic response[76]. While guidelines support the role of NAC before radical cystectomy for MIBC, there remains a role for risk-stratified NAC patient selection. Clinicopatho- logic risk factors have been implemented in predicting response to cisplatin-based NAC[77,78], but efforts are underway to profile tissue-based biomarkers for this purpose. The Southwest Oncology Group (SWOG) 1314 trial prospectively profiled the ability of the COXEN tissue-based genetic classifier to predict complete patho- logic response to cisplatin-based NAC (Table 2)[79]. 252 SIUJ • Volume 3, Number 4 • July 2022 SIUJ.ORG REVIEW Recently reported results indicate limited predictive capacity of the genomic classier for individual treatment response, underscoring the importance of prospec- tive validation of predictive markers in the clinical trial setting. Our group, among others, has also been involved in profiling immunohistochemical signatures in TURBT specimens predictive of response to NAC to improve appropriate stratification of patients for NAC or upfront cystectomy[80,81]. Lastly, biomarkers could significantly aid in the ability to accurately predict and assess a complete clinical response to NAC in those electing for bladder perseveration, a concept with supporting clinical data[82] and currently being evalu- ated in 2 randomized trials (RETAIN, Alliance A031701; Table 2). Conclusions There are no currently FDA-approved predictive biomarkers for therapies in BC–neither for NMIBC nor for MIBC. The lack of clinically available predic- tive biomarkers is likely multifactorial, including diffi- culties in profiling intratumoral heterogeneity and dynamic cellular processes (ie, epithelial-mesenchy- mal transition, cell growth and proliferation, immune response) as well as the lack of “fit for purpose” profil- ing of biomarkers with mechanistic rationale. The ever-evolving armamentarium of therapeutic options further emphasizes unique unmet needs for predic- tive biomarker development. 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