Current and Emerging Strategies to Treat Urothelial Carcinoma

Simple Summary Urothelial cell carcinoma (UCC) is the ninth most common cancer worldwide and in the US the fourth most common cancer, with ~82,000 new cases (~62,000 men) diagnosed annually leading to ~17,000 deaths/year (~12,000 men). While early-stage cases exhibit more favorable outcomes, the emergence of drug resistance and distant metastasis reduces median overall survival (OS) to 12–15 months. The development of modern genetic and molecular assays to detect high-risk mutations has improved the detection of high-risk disease. Recently, immune therapies have been developed; these demonstrate markedly improved OS rates compared to treatment with chemotherapy alone. However, challenges persist and there remains an urgent, unmet need to develop and advance novel molecular and therapeutic strategies that prevent or overcome drug resistance, to improve patient outcome. Here, we provide an overview of the etiology, diagnostic approach and emerging therapeutic strategies for improving UCC patient quality of life and OS. Abstract Urothelial cell carcinoma (UCC, bladder cancer, BC) remains a difficult-to-treat malignancy with a rising incidence worldwide. In the U.S., UCC is the sixth most incident neoplasm and ~90% of diagnoses are made in those >55 years of age; it is ~four times more commonly observed in men than women. The most important risk factor for developing BC is tobacco smoking, which accounts for ~50% of cases, followed by occupational exposure to aromatic amines and ionizing radiation. The standard of care for advanced UCC includes platinum-based chemotherapy and programmed cell death (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors, administered as frontline, second-line, or maintenance therapy. UCC remains generally incurable and is associated with intrinsic and acquired drug and immune resistance. UCC is lethal in the metastatic state and characterized by genomic instability, high PD-L1 expression, DNA damage-response mutations, and a high tumor mutational burden. Although immune checkpoint inhibitors (ICIs) achieve long-term durable responses in other cancers, their ability to achieve similar results with metastatic UCC (mUCC) is not as well-defined. Here, we discuss therapies to improve UCC management and how comprehensive tumor profiling can identify actionable biomarkers and eventually fulfill the promise of precision medicine for UCC patients.


Introduction 1.Overview
Urothelial cell carcinoma (UCC, formerly known as transitional cell carcinoma, TCC) is the most common neoplasm of the urinary system.UCC is the most common histologic type of BC and accounts for ~90% of all BC [1][2][3][4].A number of histologic variants of UCC have been identified, including micropapillary, microcystic, nested, lymphoepitheliomalike, plasmacytoid, sarcomatoid, giant cell, poorly differentiated, lipid rich, clear cell and urothelial carcinoma with divergent differentiation.UCC is defined as the invasion of the cancer cells' basement membrane or lamina propria or deeper by neoplastic cells of urothelial origin.Invasion is defined as 'micro invasion' when the depth of invasion is 2 mm or less [2].The World Health Organization (WHO) classifies BCs based on differentiation as low grade (grades 1 and 2) or high grade (grade 3) [1].Distinction between low-grade and high-grade urothelial disease has implications related to risk stratification, patient management and treatment outcome.
Urothelial tumors arise and evolve through divergent genetic and phenotypic pathways [2].The advent of next-generation sequencing has allowed widespread comprehensive molecular characterization of urothelial tumors and, subsequently, the development of therapies targeting specific molecular pathways implicated in carcinogenesis, e.g., FGFR inhibition, Nectin-4, Trop-2, and HER2 targeting.As these therapies are effective in a second-line setting, they will be advanced in the treatment paradigm to localized and even non-muscle invasive disease.Some tumors progress from urothelial hyperplasia to lowgrade, non-invasive superficial papillary tumors.More aggressive variants arise from flat, high-grade carcinoma in situ and progress to invasive tumors or arise de novo as invasive tumors [3][4][5].These distinct phenotypic variants of urothelial tumors exhibit drastically different biological behavior, responses to treatment and prognoses for patients.The lowgrade papillary variant is often multifocal and tends to recur, and infrequently progresses to the muscle invasive stage.In contrast, most invasive variants develop into incurable metastases despite radical cystectomy (RC) and additional modalities.It is evident that UCC variants harbor distinct genetic defects that impact growth control, metastatic potential and treatment decisions, as in Figure 1 in [6].Low-grade, non-invasive papillary tumors are frequently characterized by activating mutations in HRAS and the fibroblast growth factor receptor 3 gene (FGFR3) [7].High-grade invasive tumors are characterized by structural and functional defects in the p53 and retinoblastoma protein (Rb) tumor-suppressor pathways [7,8].

Environmental and Hereditary Risk Factors for Urothelial Cancers
Risk factors for UCC are largely environmental, with smoking by far the most important.Smoking is a major risk factor for BC and accounts for ~50% of cases in both men and women.Current smokers are 4-5 times more likely to develop BC than non-smokers [9,10].Other environmental risk factors include occupational exposure, radiation, drinking water contaminants, and chronic bladder infections [11].These factors contribute to an increased risk of developing BC, highlighting the importance of preventative measures, e.g., smoking cessation, and minimizing exposure to harmful substances.The consequences of these risk factors and exposures leads to field changes within the urothelial tract that predispose individuals to the development of recurrent tumors, occurring in new locations in the urothelial tract, a phenomenon referred to as polychronotropism [12].Genetics are also implicated in an individual's susceptibility to urothelial BC.Studies have found that individuals with a family history of UCC have a higher relative risk of developing the disease.However, an increased risk cannot be entirely attributed to shared environmental exposures, indicating that genetic factors may play a role in BC development [13].Hereditary non-polyposis colon cancer syndrome (HNPCC) is caused by mutations in DNA mismatch repair genes and can be identified through microsatellite instability (MSI) or the absence of the corresponding protein by immunostaining.HNPCC is linked to a greater risk of developing BC and upper urinary tract/urothelial cancers (UTUC).

Novel Approaches for Urothelial Cancers
While clinical management of early-stage UCC has seen significant improvements in the past decade, treatment of locally advanced and mUCC remains difficult [14].Recently, a number of strategies and therapies have been developed for the treatment of advanced UCC.Noteworthy among these are immune checkpoint inhibitors (ICIs) and antibody-drug conjugates (ADCs) [15].More traditional approaches, e.g., radiotherapy (RT), as part of trimodal therapy, is an attractive alternative treatment in patients with urothelial muscle-invasive BC (MIBC) [16,17].Moreover, there is some evidence to suggest the effects of ICIs and RT are potentially synergistic.Photodynamic therapy (PDT) also has merit and is a promising approach.Clinical success can be limited due to the severe hypoxia which promotes ICI resistance [18].Chemotherapy and hypoxia induce programmed death ligand-1 (PD-L1) overexpression, leading to immunosuppression within the tumor microenvironment (TME).Current approaches do not thoroughly address these defects effectively by a single drug or nano-system.To address this problem, Zhou et al. developed a biomineralization method to construct MB@Bu@MnO 2 nanoparticles with a two-step oxygen regulation ability and PD-1/PD-L1 axis cascade-disruption capacity.Manganese dioxide albumin (MnO 2 @Alb) functions as the drug carrier, butformin (Bu) as a mitochondria-associated oxidative phosphorylation (OXPHOS) disruptor with PD-L1 depression and oxygen reversion ability, and methylene blue (MB) as a PDT drug with programmed cell death protein 1 (PD-1) inhibition capacity.Owing to the tumor-responsive capacity of MB@Bu@MnO 2 nanoparticles, Bu and MB were selectively delivered and released in tumors.Hypoxia was reversed by Bu inhibited oxygen consumption, and MnO 2 improved oxygen generation.Reactive oxygen species (ROS) generation was enhanced by MB@Bu@MnO 2 nanoparticle-mediated PDT owing to reversed tumor hypoxia.The immunosuppression microenvironment was reversed by MB@Bu@MnO 2 nanoparticles that enhanced immunogenic cell death and PD-1/PD-L1 axis cascade-disruption, which then promoted T-cell infiltration.Nanosystems could solve the defects of traditional PDT by disrupting the PD-1/PD-L1 axis and reversing the negative effects of hypoxia within the TME [19].

Invasive and Non-Invasive UCC
The clinical staging of BC requires a multimodal approach that combines histopathology with molecular and imaging studies [20].Most UCC are a form of non-muscle invasive BC (NMIBC).Urothelial tumors develop along two major, largely independent but somewhat overlapping biological pathways, referred to as papillary and non-papillary or solid [21].Current management of BC still relies on pathologic staging that does not always reflect the risk for an individual patient.Studies assessing molecular alterations in individual tumors are offering insights into the myriad of cellular pathways that are deregulated in bladder tumorigenesis and progression.Alterations in pathways involved in cell-cycle regulation, apoptosis, cell signaling, angiogenesis and tumor-cell invasion have been shown to influence disease behavior [22].Noninvasive UCC are further subcategorized into exophytic papillary and carcinoma in situ (CIS) based upon distinct molecular alterations.Exophytic papillary tumors, or Ta tumors, which are a type of noninvasive urothelial cancer, can have varying genetic mutations and are classified as either high or low grade [23][24][25][26][27][28][29].Low-grade Ta tumors are often associated with mutations involving receptor tyrosine kinase-Ras activation, specifically either HRAS or FGFR3 mutations, while high-grade Ta tumors are typically linked with p16INK4a homozygous deletion and a lower frequency of FGFR3 mutations [30].In contrast, CIS and invasive tumors have alterations in TP53 and RB1 [31].Muscle-invasive tumors are characterized by changes in vascular endothelial growth factors (VEGF), cadherins, and matrix metalloproteinases (MMPs) [32].

Cell Cycle Alterations
UCC is characterized by actionable, genetic alterations in specific molecular pathways that result in uncontrolled cellular proliferation, and that can be pharmacologically targeted [33][34][35].As illustrated in Table 1, genetic alterations in UCC play a pivotal role in the etiology and progression of the disease.The most well-characterized pathways in UCC are those regulating the cell cycle, particularly the p53 and RB mechanisms, which interact with mediators of apoptosis and gene regulation [7,8,29,36].Thus, TP53 is encoded on chromosome 17p13.1 and inhibits cell-cycle progression by transcriptionally activating p21WAF1/CIP1.Loss of p21 expression can predict disease progression, and its maintenance can abrogate the effects of altered TP53.Furthermore, TP53 mutations result in p53 inactivation and the rate of TP53 alterations in primary tumors increases progressively from normal urothelium to non-muscle-invasive tumors, to muscle-invasive disease and metastatic nodes [37]; TP53 is genetically altered in 49-54% of cases, while MDM2, which participates in an autoregulatory feedback loop with p53, is genetically altered in 9-11% of cases [34,38].As a prognostic, TP53 is not clinically established but has been shown to predict recurrence and cancer-specific mortality in muscle-invasive disease.Rb is a cell-cycle regulatory protein that is encoded by RB1 and that interacts with CDKs and E2F, leading to a transcription of genes required for DNA synthesis, and inactivating RB1 mutations have been confirmed in bladder tumors [7,8,31].CDKN2A/B, the genes that encode p14 and p16, inhibiting the cyclin-dependent kinases, Cdk4 and Cdk6, are altered in 5-23% of cases of MIBC [25,39].CCND1 (genetically altered in 10-14% of cases) and CCND3 (genetically altered in 4-11% of cases) encode for cyclins that promote cell-cycle progression through interaction with Cdk4 and Cdk6.CDKN1A, a potent cyclindependent kinase inhibitor, is genetically altered in ~14% of cases.How CDK inhibitors, e.g., p21, p16, and p27, negatively regulate CDKs, acting as tumor suppressors is a complex process that involves multiple pathways leading to programmed cell death [38].Bcl-2, an antiapoptotic member of the Bcl-2 family of proteins, is associated with a poor prognosis in BC patients [37].A prior study suggested that patients treated with RT for TCC of the bladder that expressed both Bcl-2 and TP53 had worse local control and disease-free, disease-specific OS [25].CDKN2A is commonly altered in human solid tumors, but prior studies have yielded conflicting evidence regarding the association between CDKN2A genomic alterations and response to ICIs.
Patients harboring CDKN2A genomic alterations were associated with a reduced benefit from ICI therapy and were more likely to have lower PD-L1 expression in tumorinfiltrating immune cells and a less inflammatory immune-TME [39].Palbociclib, a CDK4/6 inhibitor, was evaluated in a phase II trial of patients with metastatic, platinum-refractory UC with loss of p16 and intact Rb, and was not effective since only 17% of patients achieved PFS at 4 months [40].Since DNA repair pathways are altered in UC, pre-clinical studies have demonstrated the effectiveness of the combination of CDK 4/6 inhibition with a PARP inhibitor [41].Trilaciclib, another CDK 4/6 inhibitor, is being evaluated in patients with advanced/metastatic UC receiving chemotherapy followed by avelumab, which is predicted to improve outcomes by protection from myelosuppression of chemotherapy as well as enhancing the immune tumor microenvironment by inducing a transient G1 cell-cycle arrest of hematopoietic cells (NCT04887831).CDKN2A genomic alterations were associated with a reduced benefit from ICI therapy in UC as well as changes in the tumorimmune microenvironment [42,43].
Cell-surface receptors, which play a role in transducing external signals to the nucleus and control gene expression, are deregulated in UCC tumor cells.For example, activating FGFR3 mutations are common in low-grade papillary Ta tumors and BC, leading to increased Ras-mitogen-activated protein kinase (MAPK) [42].A large cohort of patients with metastatic urothelial cancer were treated with an anti-PD-L1 agent (atezolizumab) and major determinants of clinical outcome were identified [43,44].Response to treatment was associated with the CD8 + T-effector cell phenotype, with a high neoantigen or tumor mutation burden (TMB), while lack of response was associated with a signature of transforming growth factor β (TGFβ)-signaling in fibroblasts.Other factors that impact cell signaling and gene regulation in BC include the sex hormone receptors, Janus kinase family members, and meiotic recombination 11 (MRE11) [45].Reduced expression of the estrogen receptor-β (ER-β) has been linked to better progression-free survival (PFS) rates in patients with NIBC, while higher levels have been associated with more advanced tumors.Janus kinase family members, such as STAT3, can predict recurrence and survival in BC patients [46][47][48].

Influence of Angiogenesis on Invasion and Metastasis
Angiogenesis is influenced by factors secreted from tumor cells that interact with endothelial cells in the stroma.VEGFs, urokinase-type plasminogen activator (uPA), and thrombospondin 1 (TSP-1) all play a role in angiogenesis and UCC progression.Elevated levels of VEGFs and uPA have been associated with poor clinical outcomes, while TSP-1 acts as an inhibitor of angiogenesis and under expression is linked to reduced OS [47,[49][50][51][52][53][54].Reduced expression of E-cadherin has been linked to tumor recurrence and disease progression, and poor OS in BC patients [55].Several protease families, including matrix metalloproteinases (MMPs), can also modulate the tumor's ability to disrupt the extracellular matrix and invade neighboring tissue.Overexpression of MMP-2 and MMP-9 is associated with advanced-stage BC and worse OS [56,57].

Effect of Hypoxia on UCC Invasion and Metastasis
A major aspect in the development of various tumors, including UCC, is growth under hypoxic or/and normoxic conditions that is orchestrated by activated signaling through two major transcription factors, hypoxia-inducible factor 1-alpha (HIF-1α) and HIF-2 [58][59][60].High immunohistochemical (IHC) expression of HIF-1α in primary UC tumors is associated with higher-grade disease, vascular endothelial growth factor-related angiogenesis, and worse prognosis with regard to disease-free and overall survival in both superficial and invasive disease.Hypoxia-induced autophagy may also propagate chemoresistance to cisplatin via the HIF-1α pathway.Resistance to ICIs presents a major obstacle in mUCC treatment and the clinical importance of hypoxia and immune status has been recently recognized [59].The hypoxic tumor microenvironment prevents sufficient glucose oxidation, induces oxidative phosphorylation, and promotes the accumulation of lactic acid and adenosine [59][60][61].These metabolic products lead to disruptions in T-cell ratios, T-cell inactivation, recruits immunosuppressive Tregs and decreases the invasion of antigen-presenting monocytes and dendritic cells to impair the efficacy of ICIs [62][63][64].

Immune Dysregulation in UCC
UCC is characterized by extensive mutational heterogeneity, which is detectable even in early-stage disease and sets the stage for the evolution of resistance [65,66].The emergence of treatment-resistant clones is a critical barrier to cure in patients with UCC.Chemotherapy and immunotherapy both act as selective pressures that shape the evolutionary trajectory of UCC throughout the disease course.PD-L1 is a checkpoint protein that impedes immune function, allowing tumor cells to proliferate, and PD-L1 expression on tumors has been linked to advanced stage and grade, disease progression, and worse OS after cystectomy [67,68].Tumor cells avoid immunity through immune escape, upregulation of immunosuppressive molecules, recruitment of immunosuppressive cells within the TME, and secretion of immunosuppressive signaling molecules, e.g., cytokines.Cisplatin-based chemotherapy has been the standard of care for UCC for three decades.Resistance to PD-1/PD-L1 blockade remains a significant challenge and limitation to clinical application.Defects in the antigen presentation machinery, lack of tumor antigen, T-cell dysfunction, PD-L1 negative tumor cells, non-coding RNAs that regulate tumor immunity, myeloid-derived suppressor cells (MDSCs) activity and immune checkpoints, and the gut microbiome have been implicated in patient response to anti-PD-1/PD-L1 therapy [69][70][71][72].As a consequence, ICIs have been approved to treat advanced or metastatic BC unreponsive to Bacillus Calmette-Guerin (BCG).

Screening, Diagnostic Approach and Staging of Urothelial Carcinoma
Hematuria is the most common symptom in patients with urothelial cancer.Irritative voiding symptoms, such as dysuria, may indicate carcinoma in situ (CIS) or a bladder tumor in at-risk patients, e.g., smokers.Patients are risk-stratified, and intermediateand high-risk patients are recommended to undergo cystoscopy to confirm the diagnosis and obtain a biopsy [73].The staging of UCC presents a major challenge as the depth of invasion determined by cystoscopy and Transurethral Resection of Bladder Tumor (TURBT) do not uniformly correlate [74].Although a TURBT specimen can confirm the presence of muscle-invasive (T2) disease, it cannot provide information on more advanced invasion due to the risk of bladder perforation.Where suboptimal TURBT is performed, there will be detrimental consequences on patient outcomes in regard to undergrading or understaging, increased recurrence or progression.A TURBT specimen should contain muscularis propria, but if absent, a repeated TURBT is often recommended.CT or noninvasive MRI can detect extravesical or nodal disease, and is more reliable if performed with a distended bladder before transurethral resection.FDG-PET/CT imaging may aid in the staging of muscle-invasive disease and detecting metastatic BC [75].However, its usefulness in local staging is limited by the urinary excretion of FDG [75,76].For non-muscle-invasive tumors, histologic grading is more relevant since almost all muscleinvasive neoplasms are high grade.Although there is increasing interest in utilizing molecular detection of circulating tumor cells (CTCs) in patients with UCC, the reported diagnostic effectiveness of these methods has been inconsistent.CTC detection assays for UC have low diagnostic sensitivity but high specificity for UC diagnosis.The timing of disease assessment is a crucial consideration in CTC detection since surgical interventions may result in a temporary dissemination of CTCs in the bloodstream, while chemotherapy and other systemic treatments may destroy CTCs or reduce the expression of markers, leading to a conversion of CTC-positive patients to CTC-negative [77].Additional studies are needed to standardize techniques and determine the best marker combinations for detecting CTCs in UCC and BC patients.

Initial Treatment
Initial treatment and prognosis of patients diagnosed with UCC depends on key factors including anatomical site, extent (stage), and histological grade.NMIBC, with the exception of CIS, can be treated by transurethral resection leading to excellent survival outcomes.One successful way of treating NMIBCs is using BCG vaccine intravesical immunotherapy, which decreases the risk of recurrence and progression of NMIBCs [78] Intrauterine injection of BCG causes extensive inflammation in the bladder wall, which targets tumor cells, but BCG intravesical immunotherapy may have short-term immune-stimulating effects [79].MIBC and UTUC often require RC and/or nephroureterectomy (RNU) [80].Systemic chemotherapy, that consists of a cisplatin-based regimen for mUCC, generally does not achieve durable responses.Therefore, the treatment outcome of patients with mUCC has been poor, with an OS rate of ~15%.Cisplatin-based neoadjuvant chemotherapy (NAC) is the standard treatment for MIBC, with benefits including downstaging and elimination of micrometastatic disease before RC [81,82].However, patient eligibility for cisplatin-based NAC is limited by a range of factors, e.g., ECOG, creatinine clearance with hearing loss, neuropathy, and heart failure [83,84].Tri-modality treatment therapy (TMT) involving TURBT, radiation therapy, and concurrent chemotherapy, is a commonly used alternative.In clinical trials, TMT has demonstrated 5-year OS rates comparable to NAC with RC, which yields rates of 48-65% [85].MIBC is associated with a higher incidence of distant metastasis compared with NMIBC.

Bladder Preservation in UCC
TMT has demonstrated safety and efficacy in multiple studies [86][87][88].Bladder preservation therapies, e.g., partial cystectomy TMT, are worth consideration for MIBC patients unfit for RC, ineligible for chemotherapy, or hesitant to undergo the procedure [84].Recent studies have shown that TMT may have better cancer-specific survival and OS than RC [84][85][86][87][88][89][90].Studies are ongoing to explore bladder preservation options for non-muscleinvasive disease (pT2 or lower) and in patients that achieve a complete response (CR) to NAC.Tumor genomic profiling technology may help to identify biomarkers that can predict response to NAC and provide treatment guidelines [91][92][93].Clinical trials, e.g., RETAIN, are exploring active surveillance and non-surgical local therapy options for MIBC patients with specific genetic alterations and <cT1 disease on restaging TURBT [94,95].

Surgical
Management of UCC relies heavily on surgical intervention, particularly for earlystage disease.TURBT is the most frequently employed surgical technique for NMIBC and for patients eligible for bladder preservation therapy [91].In more advanced cases, RC may be required [96,97].Recent advances in surgical methods have given rise to minimally invasive approaches, e.g., robotic-assisted surgery [98,99].Moreover, ongoing research efforts aim to develop novel surgical modalities, such as photodynamic therapy (PDT), which employs light to activate a photosensitizing agent and selectively destroy malignant cells while sparing healthy tissue [100].

Single Agent and Combination Chemotherapy
Patients with mUCC can benefit from a variety of chemotherapy combinations, e.g., MVAC and gemcitabine plus cisplatin [101,102].In certain cases where combination chemotherapy is not suitable or prior treatment has failed, single-agent chemotherapy options, e.g., platinum compounds (cisplatin, carboplatin), gemcitabine, vinca alkaloids (vinblastine, vinflunine), anthracyclines (doxorubicin, epirubicin), methotrexate, taxanes (paclitaxel, docetaxel, and nanoparticle albumin-bound paclitaxel-nabpaclitaxel), and ifosfamide may be used [103,104].However, the response to single-agent chemotherapy is brief, and there is no evidence showing improvement in survival with first-line therapy.
Worldwide, ~75% of newly diagnosed UC cases are NMIBC and the current gold standard for treatment is surgical removal by TUR.Due to the high rate (~70%) of recurrence after TUR, patients require an intensive follow-up regime that lasts many years following the initial diagnosis.This lifelong requirement for disease surveillance means UC is associated with the highest cost from diagnosis to death [38,105].Urine cytology has high sensitivity in detecting high-grade urothelial tumors (84%), but low sensitivity in low-grade tumors (16%).The FDA has now approved six urinary assays for clinical in vitro diagnostic use: BTA-stat, BTA-TRAK, NMP22, NMP22 BladderChek, ImmunoCyt/uCyt+, and UroVysion fluorescence in situ hybridization (FISH).These tests show high sensitivity in the detection of high-grade and late-stage UC, but are unable to detect low-grade malignancies and tend to give false positive results for benign inflammatory conditions.As such, they cannot be used as stand-alone diagnostic tests for UC.Newer tests and biomarkers to stratify treatment are needed.Prior studies analyzed 118, 187 deidentified tumor samples and identified PDL1 amplifications in 843 (0.7%), including more than 100 types of solid tumors.Most PDL1-amplified tumors (84.8%) had a low to intermediate TMB.PDL1 amplification did not always correlate with high-positive PD-L1 expression by immunohistochemical analysis.

Targeting FGFR
Erdafitinib is a potent FGFR1-4 tyrosine kinase inhibitor.FGFRs are essential in regulating cell proliferation, migration, differentiation, and survival [106].Up to 20% of patients with advanced UCC have FGFR alterations, and such mutations are even more frequent (37%) in patients with upper-tract urothelial carcinoma.Hence, FGFR-inhibition may be appropriate in patients with luminal I subtype disease, in which immunotherapeutic approaches may be less effective.Erdafitinib has demonstrated activity among patients with locally advanced and unresectable mUCC and with specific FGFR alterations.Erdafitinib has demonstrated activity among patients with locally advanced and unresectable mUCC and with specific FGFR alterations.In an open-label, phase 2 study, 99 patients with locally advanced or mUCC and FGFR alterations who had disease progression after previous chemotherapy, were enrolled.Patients were randomly assigned to receive erdafitinib in either an intermittent or a continuous regimen receiving a median of five cycles.The confirmed response rate was 40%, with a median duration of PFS of 5.5 months and OS of 13.8 months.Responses were prompt and not influenced by the number or type of prior treatments, tumor location or presence of visceral metastasis.Treatment-related adverse events (TRAEs) of ≥grade 3 were reported in 46% of patients, with 13% of patients discontinuing treatment.Common side effects included diarrhea, nausea, vomiting, fatigue, and rash, while more serious AEs were hyperphosphatemia and detachment of retinal pigment epithelium.Table 2 provides a comprehensive overview of completed and ongoing clinical trials, shedding light on the evolving landscape of treatment strategies for UCC.

Immune Checkpoint Inhibitors
Immunotherapy and targeted treatments are options to address situations where BCG has been unsuccessful, and for cancers that have progressed following cisplatin-based therapies (see Table 3) [107].High TMB, DNA damage-response mutations, the presence of genomic instability and high PD-L1 expression make UCC suitable for immunotherapy.The treatment landscape for patients with advanced UCC has been significantly changed with the approval of ICIs, e.g., atezolizumab, pembrolizumab, avelumab, and durvalumab.These approvals have opened new treatment options for patients with disease progression after platinum-based chemotherapy, those who are not eligible for first-line cisplatin-based therapy and have PD-L1-positive tumors, and those who are not suitable candidates for platinum-based therapy [108,109].In 2017, pembrolizumab, a humanized monoclonal antibody targeting PD-1, was approved for patients with advanced UCC who had progressed after cisplatin-based therapy.A phase 3 randomized, controlled trial included 542 patients with advanced UCC randomly assigned to receive pembrolizumab or the investigator's choice of chemotherapy.The co-primary endpoints were OS and PFS.Results showed that pembrolizumab improved OS compared to chemotherapy, with a median OS of 10.3 months in the pembrolizumab group vs. 7.4 months in the chemotherapy group.The study also found fewer TRAEs of any grade in the pembrolizumab group than the chemotherapy group [110].Interestingly, patients benefited from pembrolizumab regardless of PD-L1 expression, measured as the combined positive score (CPS), defined as the proportion of PD-L1-expressing tumor and infiltrating immune cells relative to the total number of tumor cells.PD-L1 positivity was defined as having a CPS ≥10%.Avelumab, another ICI, received approval in 2020 as maintenance therapy for patients with locally advanced or mUCC who did not progress after first-line platinum-based chemotherapy.The results of a phase 3 trial showed that the addition of maintenance avelumab, an anti-programmed death-ligand 1 (PD-L1) monoclonal antibody, to best supportive care, prolonged OS in patients with unresectable locally advanced or mUCC who did not have disease progression with first-line chemotherapy.The study enrolled 700 patients, and OS at 1 year was significantly better in the avelumab group compared to the control group.Avelumab also significantly prolonged OS and PFS in the PD-L1positive population.The incidence of AEs was higher in the avelumab group but generally manageable [111].Following demonstration of efficacy, avelumab was incorporated as a treatment option for patients with advanced UCC.
After the success of ICIs in treating mUCC, they have been introduced as adjuvant therapy.In 2021, the FDA approved nivolumab for patients who have undergone complete resection and have high-risk UCC, defined as residual cancer of ≥pT2 or pN+ after receiving neoadjuvant cisplatin-based chemotherapy or ≥pT3 or pN+ without prior chemotherapy.A phase 3, multicenter, double-blind, randomized, controlled trial compared the efficacy of nivolumab vs. placebo in 709 patients with muscle-invasive UCC who had undergone radical surgery.The study showed that nivolumab significantly improved disease-free survival and survival free from recurrence outside the urothelial tract, especially in patients with a tumor PD-L1 expression level of >1% [112].Atezolizumab, previously used in the treatment of mUCC [113], has been discontinued recently based on a phase III trial which demonstrated that, although the addition of atezlizumab to platinum-based chemotherapy resulted in improved PFS, it did not lead to any OS advantages.Consequently, the FDA revoked its regulatory approval and it is no longer used.Despite progress in our understanding of the molecular basis of UCC, the heterogeneity of individual tumors and evolutionary pressures imposed by therapy have hampered the ability to effectively eradicate and control the disease [114,115].Advances in our understanding of molecular mechanisms of tumorigenesis have translated into knowledge-based therapies directed against specific oncogenic signaling targets and fundamental cellular processes [116].Recently, it was revealed that mitochondria oxidative phosphorylation (OXPHOS) depression can be used as an effective PD-L1 downregulation method.In this method, IR-LND is prepared by conjugating mitochondria-targeted heptamethine cyanine dye IR-68 with mitochondrial complexes I and II depression agent lonidamine (LND), which further self-assembles with albumin (Alb) to form IR-LND@Alb nanoparticles.The PD-L1 expression in tumors is selectively and effectively depressed by IR-LND@Alb nanoparticles.The results suggest that the antitumor efficacy of PD-L1 depression may be superior to conventional anti-PD-L1 antibodies [19,108].

Antibody-Drug Conjugates
Two antibody-drug conjugates (ADCs) have been approved for the treatment of BC.Enfortumab vedotin targets Nectin-4, which is overexpressed in numerous BCs and is linked to the microtubule inhibitor conjugate monomethyl auristatin E [117,118].Enfortumab vedotin was evaluated in a global, open-label, phase 3 trial that enrolled 608 patients who had previously received platinum-containing chemotherapy and PD-1/PD-L1 inhibitors [119].Patients were randomly assigned to receive either enfortumab vedotin or chemotherapy.Results showed that OS was longer in the enfortumab vedotin group than the chemotherapy group, with a median OS of 12.9 vs. 9.0 months, respectively.PFS was longer in the enfortumab vedotin group, with a median PFS of 5.6 months compared to 3.7 months in the chemotherapy group.The incidence of TRAEs was similar in both groups.Subsequently, it was approved by the FDA.Although skin reactions, peripheral neuropathy, and hyperglycemia are common side effects, they are mostly mild to moderate in severity.Sacituzumab govitecan is a new type of ADC that targets Trop-2 through an anti-Trop-2 humanized monoclonal antibody hRS7 IgG1κ combined with SN-38, which is the active metabolite of the topoisomerase 1 inhibitor irinotecan [120,121].It is recognized for its distinctive toxicity profile that may lead to diarrhea, nausea, vomiting, fatigue, neutropenia, and even severe or life-threatening infusion reactions.The approval of enfortumab vedotin and sacituzumab govitecan was a significant milestone in the treatment of UCC.

Cellular Vaccines and Oncolytic Viruses
Vaccine therapy activates the immune system to target cancer cells using various methods, such as synthetic peptides and viral vectors.In particular, PANVAC-VF is an example of a vaccine therapy that has shown positive results in clinical trials [122], but further research is needed to optimize its dosing and effectiveness in combination with other treatments.Oncolytic viruses selectively replicate within tumor cells, resulting in their destruction and the release of additional oncolytic viruses and tumor antigens.For instance, CG0070 is an example of an oncolytic virus that specifically targets cells with RB gene mutations, which are common in UCC [123].Despite showing promising results, the efficacy of vaccine therapy and oncolytic viruses in combination with other therapies needs further investigation.

CAR-T Therapy
CAR-T therapy is an emerging immunotherapy approach that involves genetically modifying a patient's T-cells to target and attack cancer cells.In UCC, CAR-T therapy is being investigated as a potential treatment for patients with advanced or metastatic disease who have failed traditional therapies [124,125].While still in early clinical trials, CAR-T therapy has shown improving responses in some patients with UCC.However, more research is needed to optimize the therapy and determine its long-term safety and efficacy.

Antiangiogenics
There is currently no established role for antiangiogenic agents in the treatment of advanced or metastatic BC.Ramucirumab and bevacizumab, both VEGF pathway inhibitors, have shown improved PFS but not OS in clinical trials [130,131].Cabozantinib, a multi-kinase inhibitor including VEGF receptors, is an investigational agent for platinumrefractory mUCC as a single agent or in combination with ICIs, with promising objective response rates [132].

Herbal Medicines
There is accumulating, emerging information to suggest that herbal medicines could have a beneficial effect on cancer therapy and/or treatment-induced adverse side effects.Historically, natural plant-derived drugs have been considered for cancer treatment.Sanguinarine (SANG), a naturally isolated plant alkaloidal agent, possesses chemopreventive effects [133].It has been reported that SANG impedes tumor metastasis and development by disrupting a wide range of cell signaling pathways and its molecular targets, e.g., BCL-2, MAPKs, Akt, NF-κB, ROS, and microRNAs.Additional preclinical trials may be required before these agents advance to the clinical stage.

Molecular Classification as a Prognostic to Guide Treatment Decisions
UCC are biologically, molecularly and clinically heterogeneous tumors and clinicopathological factors fail to consistently predict treatment response and patient outcome.Molecular classifiers may help organize patients into less heterogeneous subclasses and stratify treatment decisions.Muscle-invasive BC (MIBC) is a molecularly diverse disease with heterogeneous clinical outcomes [23,[134][135][136][137][138].Table 4 lists a number of biomarkers that may have value in stratifying patient treatment decisions.Recent large-scale transcriptomic profiling studies of MIBC tumors have revealed molecularly distinct clusters.Biomarkers that are prognostic and/or predictive of therapeutic responses can be tested and validated to stratify patient treatment.Several molecular classifications have been proposed, but the molecular, genetic and histologic diversity of the disease impedes clinical application.To achieve an international consensus on MIBC molecular subtypes that reconciles the published classification schemes, the Bladder Cancer Molecular Taxonomy Group developed a classification system using transcriptomes from 1750 patients and a network-based analysis of multiple independent classification systems [24].The primary molecular classification subdivisions were basal and luminal subtypes, and these subtypes were further subdivided by TCGA into squamous, infiltrated, luminal-papillary, luminal/genomically unstable (GU), and neuronal/small cell carcinoma (SCC) subtypes [23,24,[134][135][136][137][138][139].Each subtype is associated with unique biologic, histologic and genetic characteristics.Hence, treatment strategies may be guided by oncogenic mechanisms, immune and stromal cell infiltration, and histologic and clinical features.This TCGA analysis provided a platform to develop and guide therapies for stratified MIBC patient populations (Figure 1).Additional, prospective TCGA-based analyses are needed to address NMIBC and metastatic disease.In addition, the patient populations used to derive the current primary molecular classification were predominantly of European ancestry and it remains uncertain whether these results are applicable to other ancestral groups.The impact of ethnicity and socioeconomics may impact the biology of the disease.Prospective clinical validation of biomarkers for the six molecular subtypes could guide treatment decision-making.In addition, biomarkers within the tumor, immune cells, adjacent lymph nodes, and the TME could impact patient stratification and treatment decisions for the use of targeted therapies, ICIs, ADCs, cancer vaccines and cellular therapies.Using patient tumor biopsies, genomic DNA was isolated and assayed using whole-exome sequencing (WES) and targeted next-generation sequencing (NGS) techniques to assess for APOBEC mutational signature.APOBEC-high are more likely to have mutations in DNA damage response genes (TP53, ATR, BRCA2) and chromatin regulatory genes (ARID1A, MLL, MLL3), potentially leading to a hypermutation phenotype and subsequent enhanced immune response against the tumor.IMvigor-130 trial-mUC patients with APOBEC mutational signature had significantly higher TMB and improved OS with atezolizumab containing regimens in the first-line cisplatin-ineligible scenario [170] Immunotherapy Markers

PD-L1-IHC 40 patients 20-72 Locally advanced and mUCC
Patient tumor biopsies were assayed for PDL1 expression using immunohistochemistry (IHC) and expressed as a combined positive score (CPS, positive tumor cells and immune cells divided by the total number of cells).PD-L1 expression and high (TMB) may predict better responses to ICIs, but patients without these biomarkers may still respond to immunotherapy.Additional caveats include a lack of standardization, tumor heterogeneity and other factors influencing the TME.

Locally advanced and mUCC
Retrospective pan-cancer analysis of PD-L1 immunohistochemistry and gene amplification, tumor mutation burden and microsatellite instability in 48,782 cases.analysis of all cases in which both PD-L1 IHC (using the DAKO 22C3 IHC assay with either tumor proportion score (TPS) or combined positive score (CPS); or the VENTANA SP142 assay with infiltrating immune cell score (IC)) and comprehensive genomic profiling (CGP) were tested at Foundation Medicine between January 2016 and November 2019.PD-L1 positivity was defined per the CDx indication and tumor proportion score (TPS ≥ 1) for indications without a CDx claim; and TMB positivity is defined as ≥10 mutations/Mb.A total of 48,782 cases were tested for PD-L1 IHC and CGP.Patient tumor biopsies were assayed for PD-L1 amplification using PD-L1 RNA in situ hybridization (RNAish).PD-L1 amplification was detected in only 0.7% of solid tumors.Amplification had a low correlation with PD-L1 IHC and did not correlate with TMB.Chemotherapy-resistant UCC lacks a uniformly curative therapy, and deciphering how chemotherapy directs selective pressure is a central question with clinical implications [139,177].Therefore, to identify potential markers that correlated with treatment response, the genetic landscape of chemoradiation-treated UCC samples was analyzed [139].Whole-exome sequencing (WES) and clonality analysis of 72 UCC samples was performed, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy.Results showed that chemotherapy-treated UCC is characterized by intra-patient mutational heterogeneity, and that the majority of mutations were not shared.Mutation profiling between primary and recurrent tumors suggested that pre-existing, resistant clonal populations represented the primary mechanism of chemoradiation treatment failure.Cisplatin-based chemotherapy remains the standard of care for patients with muscle-invasive UCC but molecular determinants of response remain incompletely understood [143].Whole-exome-sequencing was performed on pretreatment tumors and germline DNA was obtained from 50 patients with MIBC who had received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis "responders," 25 pT2+ "nonresponders") to identify mutations  Chemotherapy-resistant UCC lacks a uniformly curative therapy, and deciphering how chemotherapy directs selective pressure is a central question with clinical implications [139,177].Therefore, to identify potential markers that correlated with treatment response, the genetic landscape of chemoradiation-treated UCC samples was analyzed [139].Whole-exome sequencing (WES) and clonality analysis of 72 UCC samples was performed, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy.Results showed that chemotherapy-treated UCC is characterized by intra-patient mutational heterogeneity, and that the majority of mutations were not shared.Mutation profiling between primary and recurrent tumors suggested that pre-existing, resistant clonal populations represented the primary mechanism of chemoradiation treatment failure.Cisplatin-based chemotherapy remains the standard of care for patients with muscle-invasive UCC but molecular determinants of response remain incompletely understood [143].Whole-exome-sequencing was performed on pretreatment tumors and germline DNA was obtained from 50 patients with MIBC who had received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis "responders," 25 pT2+ "nonresponders") to identify mutations that had specifically occurred in responders.ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q < 0.01).Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2.Somatic ERCC2 mutations were shown to correlate with complete responses to cisplatin-based chemosensitivity in muscle-invasive UCC.Inflammation-driven phenotypic plasticity also alters the antigenic landscape of tumor cells, rewiring oncogenic signaling networks to reprogram immune cell functions [178].
A systematic meta-analysis merged gene expression datasets from the GEO repository for 18 datasets and identified 815 differentially expressed genes (DEGs) [171].The key hub genes were screened for their differential expression in patient urine and blood plasma samples.A three-gene signature model, including COL3A1, FOXM1, and PLK4, was built.The predictive value regarding muscle-invasive patients' responses to neoadjuvant chemotherapy was analyzed and a six-gene signature model, including ANXA5, CD44, NCAM1, SPP1, CDCA8, and KIF14, was developed.The study identified nine key biomarker genes-ANXA5, CDT1, COL3A1, SPP1, VEGFA, CDCA8, HJURP, TOP2A, and COL6A1-which were differentially expressed in urine or blood, held a prognostic or predictive value, and were immunohistochemically validated.Promising biomarkers may eventually lead to greater personalization of care for patients with BC, but remain investigational.
Five programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors are currently approved for treatment of locally advanced or mUCC of the bladder and the upper urinary tract [172].Due to restrictions by the FDA and EMA, first-line treatment with Atezolizumab and Pembrolizumab in platinum-ineligible patients requires immunohistochemical PD-L1 testing.In the second-line setting, all drugs are approved without PD-L1 testing.The balance of adaptive immunity and pro-tumorigenic inflammation in individual TME is associated with PD-1/PD-L1 resistance in UCC, with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood [173].The quantity and spatial distribution of stromal tumor-infiltrating lymphocytes (sTIL) within the TME also predict stages of tumor inflammation, subtypes, and patient survival, and correlate with expression of immune checkpoints in an analysis of 542 patients with MIBC [174].High sTILs indicated an inflamed subtype with an 80% 5-year DSS, and a lack of immune infiltrates identified an uninflamed subtype with a survival rate of less than 25%.A separate immune evading phenotype with upregulated immune checkpoints was associated with poor survival.Within the TIME are tertiary lymphoid structures (TLS), which can mediate antitumor activity via immune cells.High TLS amounts and close tumor distance correlated significantly with an inflamed phenotype and favorable survival.The uninflamed and evasion phenotypes showed lowest TLS numbers, farthest tumor distances, and shortest survival.High inflammation also correlated with increased neoantigen load and mutational burden.
There is a need in clinical practice to identify biomarkers that can serve as tools for risk assessment, screening and early detection of UCC, as well as for accurate diagnosis, patient prognosis, prediction of response to therapy, and cancer surveillance and monitoring response [175].Better biomarkers are needed as tools to identify patient subsets since precision oncology is effective only in patients with specific cancer genetic mutations.Systematic, bioinformatic tools represent a valuable approach to identify and validate novel biomarkers with greater sensitivity, specificity, and positive predictive value.Publicly available genomewide datasets, greater availability of patient pre-and post-treatment samples, analysis of tumor and immune cells, (urinary) extracellular vesicles, and the microbiome are promising sources of biomarkers.Low-or non-invasive methods of obtaining biomarkers also are appealing.Personalized treatment approaches based on individual patient characteristics and tumor biomarkers hold promise to stratify and optimize therapy and improve patient qualityof-life and OS, while reducing treatment-related toxicities.Additional candidate biomarkers to personalize BC care are under investigation, but significant obstacles must be overcome before they can be implemented in clinical practice.

Conclusions
Despite advances in diagnostic and therapeutic strategies, mUCC remains a significant challenge.The diagnostic and monitoring techniques used for urologic cancers comprise a group of invasive methodologies that still lack sensitivity and specificity [176].Early diagnosis is crucial to improve patient outcomes and current treatments offer a range of options to manage UCC and mUCC.The treatment of mUCC has shifted dramatically following the introduction of ICIs [68,179].Similarly, BCG is one of the most successful immunotherapies and remains the gold standard of care for patients with high-risk NMIBC, with initial response rates of ~70% [180].Collectively, recent trials indicate that ~30% of mUCC patients demonstrate a response to ICIs.Recently approved ICIs are available as first-line therapy for cisplatin-ineligible patients or as second-line therapy for mUCC patients.Currently, selection of patients with mUCC to receive first-line immunotherapy is informed by expression of PD-L1 [181].Patients must also be cisplatin-ineligible, but may be carboplatin-eligible if PD-L1 expression is sufficiently high.Patients with mUCC who are ineligible for any platinum-based chemotherapy may also receive immunotherapy in the first line, regardless of PD-L1 expression.High tumor mutational burden and microsatellite instability may be used to select patients for treatment with immunotherapy in cases of advanced BC, with some non-urothelial histologies in which patients have progressed.Promising innovations that may potentially advance precision medicine in UCC include multi-omic approaches, innovative trials designs, cell-free DNA, and machine learning algorithms.Biomarkers may allow for better disease classification, prognostication, and development of more efficacious noninvasive detection and surveillance strategies, as well as for the selection of therapeutic targets which can be used in BC, particularly in a neoadjuvant or adjuvant setting.Clinically relevant and useful biomarkers are also needed to identify high-risk disease, to optimize and personalize ICIs treatment for UCC.At present, alterations of FGFR2/3 are the only genetic feature of mUCC currently used to select patients for targeted therapy [140,182,183].In addition, erdafitinib is the only targeted therapy specifically approved for FGFR-altered UCC.
Recent studies have demonstrated using incorporating immunotherapies, i.e., ICIs, BCG, cytokines, adoptive T-cell immunotherapy, DCs, and macrophages, has a potential benefit for UCC patients.Nanoparticles, nano-immunotherapy, herbal medicines, and agents that overcome hypoxia-mediated immunosuppression are all potential approaches to augment PD-L1 expression on tumor cells to promote the PD-L1/PD-1 interaction.Antibiotics, fecal microbiota transplantation, and diet regulation are feasible, practical approaches to manipulate the gut microbiome and improve patient responses to immunotherapy.Prevention strategies, e.g., lifestyle modifications, smoking cessation, and chemoprevention, have the potential to reduce the incidence and improve the management and outcome of UCC.Studies that explore the utility of ICIs in the adjuvant or neoadjuvant settings, or in combination with chemotherapy, may reveal strategies to enhance patient response.The treatment paradigm for UCC is rapidly changing and novel biomarkers are emerging to select patients for appropriate therapies.Taken together, these advances offer an opportunity to improve patient survival.

Table 4 .
Cont.The proportion of VEGF+ cells were defined to calculate a proportion score.The relative intensity was quantitated into an intensity score.Finally, a total immunostaining score was determined as the product of a proportion score and intensity score.Elevated VEGF correlates with worse outcome.VEGF pathway inhibition attenuates tumor proliferation and invasion.Ramucirumab, a fully humanized monoclonal antibody that binds VEGF receptor 2, has shown benefit both in randomized phase II and III trials.Results with sunitinib, pazopanib, vandetinib, or cabozantinib were not convincing.