BCG-Unresponsive Non-Muscle-Invasive Bladder Cancer: Current Treatment Landscape and Novel Emerging Molecular Targets

Urothelial carcinoma (UC), the sixth most common cancer in Western countries, includes upper tract urothelial carcinoma (UTUC) and bladder carcinoma (BC) as the most common cancers among UCs (90–95%). BC is the most common cancer and can be a highly heterogeneous disease, including both non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) forms with different oncologic outcomes. Approximately 80% of new BC diagnoses are classified as NMIBC after the initial transurethral resection of the bladder tumor (TURBt). In this setting, intravesical instillation of Bacillus Calmette–Guerin (BCG) is the current standard treatment for intermediate- and high-risk patients. Unfortunately, recurrence occurs in 30% to 40% of patients despite adequate BCG treatment. Radical cystectomy (RC) is currently considered the standard treatment for NMIBC that does not respond to BCG. However, RC is a complex surgical procedure with a recognized high perioperative morbidity that is dependent on the patient, disease behaviors, and surgical factors and is associated with a significant impact on quality of life. Therefore, there is an unmet clinical need for alternative bladder-preserving treatments for patients who desire a bladder-sparing approach or are too frail for major surgery. In this review, we aim to present the strategies in BCG-unresponsive NMIBC, focusing on novel molecular therapeutic targets.


Introduction
Urothelial carcinoma (UC), which includes bladder carcinoma (BC) and upper urinary tract urothelial carcinoma (UTUC), is the sixth most common cancer in Western countries [1].BC, as the most common cancer, is a highly heterogeneous disease that includes both non-muscle-invasive bladder carcinoma (NMIBC) and muscle-invasive bladder carcinoma (MIBC) with different oncologic outcomes [2][3][4].Approximately 80% of newly diagnosed bladder carcinomas are classified as NMIBC after the initial transurethral resection of the bladder tumor (TURBt), representing a broad spectrum of disease [5].While low-risk NMIBC patients are mainly treated with TURBt alone, intermediate-and high-risk patients often receive adjuvant treatments to reduce disease recurrence and progression [5][6][7].In this context, intravesical administration of Bacillus Calmette-Guerin (BCG) is the current standard treatment [8].Despite adequate BCG treatment, recurrence occurs in approximately 40% of patients and MIBC in 15% [9].In particular, highly recurrent BCs represent the highest risk of disease [8].The European Association of Urology guidelines (EAU) currently define "BCG-unresponsive" as all BCG-refractory tumors and those that develop T1/Ta HG recurrence within six months of completion of adequate BCG exposure or develop carcinoma in situ (CIS) within twelve months of completion of adequate BCG exposure."Adequate BCG exposure" is defined as the completion of at least five of six doses of a first induction course plus at least two of six doses of a second induction course or two of three doses of a maintenance regimen.Patients with NMIBC who do not respond to BCG are extremely unlikely to benefit from further BCG administration and represent a patient cohort for whom new treatment options are urgently needed [10].High-risk NMIBCs who do not respond to BCG, therefore, present a therapeutic challenge.
In this specific scenario, non-surgical options are limited, and according to the EAU guidelines [5,11], the recommended treatment for BCG-unresponsive disease remains radical cystectomy (RC) and urinary diversion (UD).RC and UD remain complex major urological procedures with a recognized high perioperative morbidity due to patient, disease, and surgical factors [12,13].Numerous improvements have been made in surgical technique and perioperative management [14].However, the morbidity profile and survival after RC have remained largely unchanged [15][16][17].As this is a predominantly elderly disease, a non-negligible number of patients are considered unsuitable for such surgery [16].
Despite the unsatisfactory efficacy profile, with a complete response rate (CR) of about 18%, by 2020 the only Food and Drug Administration (FDA)-approved conservative treatment for CIS patients who did not respond to BCG was intravesical valrubicin [18].Nowadays, new therapeutic options, potentially ready for the first time, could lead to a turning point in the treatment of patients who do not respond to BCG.In such a scenario, two unmet clinical needs could emerge.First, reliable biomarkers could identify early those patients who do not respond to BCG immunotherapy.Second, the current arsenal of new intravesical and systemic treatment candidate agents could be expanded for bladdersparing strategies.If the identification of new molecular biomarkers is of interest for all disease stages of BC [19][20][21], the choice of new treatment candidates for NMIBC is wide, leading the FDA to accept single-arm clinical trials as adequate clinical evidence for evaluating therapeutics [22][23][24].The choice of new treatment candidates for NMIBC is wide, leading the FDA to accept single-arm clinical trials as adequate clinical evidence for evaluating therapeutics.
In this review, strategies for BCG-unresponsive NMIBC are presented with the aim of examining the role of alternative therapies for NMIBC, focusing on the new molecular targets.
Regarding the oncologic outcomes of intravesical gemcitabine, there is considerable heterogeneity in the assessment of its efficacy and results.In addition, there is a non-negligible discrepancy in study designs, some of which are retrospective.For example, in 2020, Hurle et al. found disease-free survival (DFS) at 12 and 24 months of 44.44% and 31.66%,respectively, in their open-label, single-arm study in which gemcitabine alone was administered to 36 patients in an induction regimen followed by maintenance therapy in responders.In this study, progression-free survival (PFS) at 12 and 24 months was 80.13% and 69.55%, respectively, while cancer-specific survival (CSS) and overall survival (OS) evaluated at 24 months were 80.68% and 77.9%, respectively [26].Skinner et al. tested gemcitabine alone in a phase II trial enrolling 58 patients and achieved a CR, i.e., negative cystoscopy, negative urine cytology, and negative biopsy, in 47% of patients at 3 months.The median recurrence-free survival (RFS) was 6.1 months and was still 21% at 24 months, with a progression rate of 36% to RC.In this study, an induction regimen was used, followed by maintenance therapy in responders [38].According to the retrospective data of Sternberg et al. on 69 patients treated with gemcitabine induction alone, the 5-year progression to MIBC rate was 19% in BCG-unresponsive patients and 22% in patients with other types of BCG failure.In this series, 27 patients achieved a CR (negative cystoscopy and urinary cytology), 19 achieved a partial response (negative cystoscopy and positive cytology), and 20 patients experienced failure (positive cystoscopy).The cancer-specific death rate was 12% in the complete responders and 18% in the remainder.Subsequent RC had to be performed on 20 patients [38].Combinations of gemcitabine with other drugs produced different outcomes.According to Chevuru and colleagues, gemcitabine plus docetaxel resulted in an RFS at 12, 24, and 60 months of 57%, 44%, and 24%, respectively, and an HG-RFS at 12, 24, and 60 months of 60%; remarkably, outcomes were slightly worse only in CIS patients compared to papillary disease-only ones [25].Similar results were found in the retrospective study by Steinberg et al. published in 2020 [27], while the same drug combination performed worse in the study by Milbar [29].De Castro reported encouraging results by combining gemcitabine with cabazitaxel and cisplatin: The triplet achieved a CR in 89% of cases with a median RFS of 27 months.RC-free survival was 94% at 1 year and 81% at 2 years [28].The combination with oral Everolimus proposed by Dalbagni resulted in an RFS of 58% at 3 months, 27% at 6 and 9 months, and 20% at 12 months [30].The association with MMC was tested by Cockerill and Lightfoot.Cockerill determined a recurrence rate of 63% with a median RFS of 15.2 months and a recurrence-free rate of 37% with a final median of 22 months after treatment [32].Lightfoot found a CR rate and 1-year and 2-year RFS of 68%, 48%, and 38%, respectively [34].
Other drugs such as paclitaxel, nab-paclitaxel [31, 40,41], valrubicin, and doxorubicin [33,46] have been studied with conflicting results.Docetaxel alone resulted in a CR in 56% to 59% of patients [36,40].In summary, despite some promising results or at least some effect in certain cases, no definitive conclusion can be drawn regarding intravesical chemotherapy after BCG administration because of the heterogeneity of the studies and protocols and the limited number of patients enrolled in most of these studies.

Chemo-Hyperthermia
The effect of hyperthermia on tumor cells has been known for decades, and several effects of hyperthermia have been described to date.First, at temperatures above 40.5 • C and above, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis is reduced, and DNA repair itself is impaired [54].In addition, hyperthermia can alter tumor blood flow in several ways.Up to a temperature of 43 • C, vasodilatation is observed, which allows better delivery of chemotherapeutic agents.Moreover, direct damage such as occlusion and destruction of the endothelial cells lining the tumor vessels, which have a reduced ability to dissipate heat compared to normal tissue, has been described, resulting in decreased blood flow to the cancer cells [55].In turn, the cancer microenvironment becomes increasingly hypoxic, and cancer cells become more sensitive to both heat and chemotherapy, while angiogenesis is inhibited via the upregulation of the plasminogen activator inhibitor-1 pathway in endothelial cells of the tumor vasculature [55].In addition, hyperthermia mimics the physiological mechanisms occurring in the immune system during fever, such as the activation of CD8 and CD4 immune cells, the infiltration of NK cells, and the production of heat shock proteins and cytokines, thus facilitating the activation of many molecular effectors of the immune system [56].In practice, an intravesical temperature ranging between 40 • C and 45 • C can be achieved either by using microwaves generated by a radiofrequency emitter incorporated in a catheter or by using a conductive system that externally heats the chemotherapeutic solution before intravesical administration.A further option is locoregional hyperthermia, which entails using an external device to generate local heating by radiofrequency [57].A scheme of the hyperthermia treatment is shown in Figure 2.
a conductive system that externally heats the chemotherapeutic solution b intravesical administration.A further option is locoregional hyperthermia, which e using an external device to generate local heating by radiofrequency [57].A scheme hyperthermia treatment is shown in Figure 2.  Previous studies focused on the synergistic effect of hyperthermia in combination with MMC, an alkylating agent administered intravesical that is more effective at high temperatures [58].Table 2 summarizes the results of studies investigating chemo-hyperthermia with MMC.Despite some discrepancies between different experiences in terms of the treatment schedule and study design, DFS reached peaks of 85% in some series [65], with most of them reporting a CR in more or less 50% of patients at 1 or even 2 years [60,64,65].Remarkably, the available data show that chemo-hyperthermia performed worse in patients with CIS [60] and in patients with papillary disease and concomitant CIS [61].In a randomized clinical trial by Tan and colleagues, a comparison between radiofrequency-induced thermochemotherapy with MMC and BCG retreatment was made, with no statistically significant differences in oncological outcomes between the two arms [61].
In conclusion, despite some interesting results, the data need to be confirmed by further studies before chemo-hyperthermia can be incorporated into routine clinical practice.

Immunotherapy and Inflammation-Targeted Agents
In the field of immunotherapy for both MIBC and UTUC, pembrolizumab has played an increasingly important role in recent years [11,66].Pembrolizumab is a humanized monoclonal antibody against programmed cell death protein 1 (PD-1), a so-called immune checkpoint inhibitor (ICI).PD-1 is a transmembrane receptor that binds to two different ligands: Programmed cell death protein 1 ligand 1 (PD-L1) and ligand 2 (PD-L2).PD-1 is expressed by activated CD4C and CD8C T cells, B cells, natural killer (NK) cells, macrophages, and dendritic cells, whereas PD-L1 is constitutively expressed on T cells, B cells, dendritic cells, regulatory T cells (Treg), monocytes, and macrophages.Binding of PD-1 to its ligands PD-L1 and 2 results in an inhibitory transduction signal for T cells.Taking advantage of this mechanism, tumor cells express PD-L1 and thus evade the immune response.By binding PD-1 and preventing interaction with its ligands, pembrolizumab may enhance the anti-tumor immune response [67].In light of this, some studies have attempted to evaluate the efficacy of pembrolizumab in BCG-refractory NMIBC.In such reports, pembrolizumab has been administered in combination with BCG by both intravesical [68] and intravenous routes and as a single agent [67].In a series of 9 patients treated with a combination of BCG and intravesical pembrolizumab, Meghani et al. reported an RFS at 3, 6, and 12 months of 100%, 67%, and 22%, respectively.In this series, PFS at 6 and 12 months was 100% and 56%, respectively, with MIBC occurrence being reported in 5 patients [69].Alanee et al. instead found a CR rate of 69% in 18 patients with BCG in combination with intravenous pembrolizumab.In this study, one patient progressed to MIBC [67].In a series of 96 patients, intravenous pembrolizumab alone resulted in a CR in 39 cases with a 51% recurrence rate, and PFS to MIBC or metastatic disease or death was 97% [10].Pembrolizumab was recently approved by the FDA for the treatment of BCG-unresponsive NMIBC.Although a phase II trial achieved CR in 41% of patients treated with pembrolizumab, less than 50% of responders continued to receive maintenance therapy [10].Table 3 lists the studies of immunotherapy for BCG-unresponsive NMIBC.A schematic summary of the immunotherapeutic approaches described is reported in Figure 3.   durable clinical activity in BCG-resistant high-risk NMIBC patients defined as high-grade Ta, T1, or CIS, tumor size greater than 4 cm, or multifocal tumors.Patients who showed CR after the induction cycle received a maintenance cycle and underwent a response assessment.CR was observed in 3 patients and was durable (≥18 months) in 2 of them.Subgroup analyses have shown transient IFN-γ and IL-6 but not TNF-α and IL-10 induction after ALT-801 administration [92].Other strategies, such as the combination of BCG with interferon (IFN)α2b, have been tested.IFNα2b is a glycoprotein produced by different cell types in response to exposure to various foreign antigens, including tumor antigens.This protein exhibits a wide range of direct and indirect anti-tumoral effects.Direct effects include inhibition of angiogenesis, cytotoxicity, and cell cycle blockage [80,81].Indirect effects include increased production of IFN-È, interleukin (IL) 12, and tumor necrosis factor (TNF)-β in BCG-stimulated cells, which may shift the polarization of the immune system toward a T-helper (Th) 1 cytotoxic response and enhance the effects of BGC itself.Despite some in vitro results, it must be acknowledged that a clear advantage over BCG alone in terms of recurrence and progression rates to date has not yet been demonstrated [82].However, DFS ranging from 42% to 53% at 24 months and up to 55% at 30 months [75,76] have been reported.
Other studies investigated the role of mycobacterium phlei cell wall-nucleic acid complex (MCNA), an immunomodulatory and antineoplastic compound derived from mycobacterium phlei that contains mycobacterial cell wall fragments complexed with biologically active nuclear acids.MCNA induces the synthesis of IL-6, IL-8, IL-12, IL-18, and TNF-α, which lead to an anticancer immune response by promoting the development of Th1 lymphocytes, stimulating the production of IFN-È, inhibiting angiogenesis, and enhancing the activity of NK and cytotoxic T cells.In addition, MCNA can selectively induce apoptosis in cancer cells via translocation of phosphatidyl serine at the cell surface, nucleosome DNA fragmentation, release of soluble protein fragments of the mitotic nuclear apparatus, and cytochrome c from mitochondria [83].A CR rate with MCNA was observed in up to 34.4% of patients at 6 months follow-up, with response rates decreasing thereafter [72,74].Of note, MCNA appeared to perform worse in CIS patients.
In 2012, Kowalski et al. tested the efficacy of oportuzumab monatox (OM), an antiepithelial cell adhesion molecule (EpCAM) single-chain humanized antibody linked to Pseudomonas exotoxin A. OM binds to EpCAM and is internalized into the cancer cell cytosol, where the toxin induces apoptosis.The authors reported a CR rate of 44%, which proved durable in 16% of the patients at 1 year.The 1-year recurrence rate was 65% [73].
Bromopirimine, an oral immunomodulatory agent, was also explored in the late 1990s.Bromopirimine displays several effects on the immune system, such as stimulating the proliferation of B cells and thus increasing serum immunoglobulin levels, enhancing macrophage-mediated and NK cell cytotoxicity, and activating T cells [84].Overall, a CR of 24% was found with an RFS ranging from 65 to 810 days in patients [79].In short, despite some interesting perspectives, clear-cut evidence for immunotherapy in BCG refractory NMIBC is still lacking.
The relationship between inflammation and tumor development has always been an important field of translational research [85].Moreover, targeting inflammation in cancer treatment, including cyclooxygenase (COX), nuclear factor-kB (NF-kB), cytokines, and/or chemokines and their receptors, represents a crucial point in tumor invasion and progression, promoting the pathway as an integral part of enhanced angiogenesis [86].Impaired standard laboratory inflammatory biomarkers have been associated with advanced disease and worse oncological outcomes [87].In particular, non-steroidal anti-inflammatory drugs (NSAIDs) are COX-competitive inhibitors used in cancer therapy and prevention [88].Recent evidence suggests that impairment of T-cell activation and cytotoxic cellular response may play a role in BCG therapy failure [89].Nogapendekin alfa-inbakicept (NAI), known as N-803, an IL-15 super agonist, is a fused protein from a human IL-15 variant bound to a dimeric human IL-15Ra domain/human IgG1 Fc.This IL-15-based immunostimulatory complex plays a crucial role in the activation and proliferation of natural killer (NK) cells as well as effector and memory T cells [90].Hypothesizing that NAI may act as this secondary activation mechanism for NK and T cells that synergistically influences BCG efficacy, Chamie et al. studied three cohorts of BCG-unresponsive patients in an open-label, multi-institutional study [68].Cohort A included patients with CIS with or without other NMIBC papillary disease treated with intravesical NAI plus BCG.Cohort C included patients treated with NAI alone.Finally, cohort B included patients with BCG-unresponsive HG Ta/T1 NMIBC who also received NAI plus BCG.The primary endpoint of the analysis was the incidence of CR at the 3-or 6-month assessment visit for cohorts A and C and the DFS rate at 12 months for cohort B. CR was achieved in 58 (71%) patients in cohort A. Specifically, the CR rates at 3, 6, and 12 months were 55%, 56%, and 45%, respectively.The median duration of CR was 26.6 months in responders.The cystectomy rate was 9% in responders.Among patients in cohort B, the DFS rate at 12 months was 55.4%, with a median DFS of 19.3 months.Of the patients who received NAI only (cohort C), CR was achieved at 3 months in only 2/10 (20%).For this reason, the recruitment of cohort C was closed following protocol-defined stopping rules.The safety profile was also evaluated: The most common treatment-emergent adverse events reported after administration of NAI plus BCG were mainly related to bladder instillation, with most of them graded 1 to 2 [68].Thus, encouraging results were obtained with a CR rate of 71% for the BCG-unresponsive NMIBC CIS cohort treated with NAI plus BCG, which was higher than the 30% proposed by Kamat et al. as a target for clinical efficacy [91].
In the same setting, ALT-801 is a recombinant humanized T-cell receptor (TCR)-IL-2 fusion protein that enhances NK and T cells' cytotoxic immune responses against p53-expressing tumor cells [86].In a phase I trial (NCT01625260), Sonpavde et al. [92] preliminarily found that intravenous infusion of ALT-801 and Gemcitabine has promised durable clinical activity in BCG-resistant high-risk NMIBC patients defined as high-grade Ta, T1, or CIS, tumor size greater than 4 cm, or multifocal tumors.Patients who showed CR after the induction cycle received a maintenance cycle and underwent a response assessment.CR was observed in 3 patients and was durable (≥18 months) in 2 of them.Subgroup analyses have shown transient IFN-γ and IL-6 but not TNF-α and IL-10 induction after ALT-801 administration [92].

Gene Therapy
Genetic instability is an essential early step in the development of BC [93].Such instability is easily detected at both the chromosomal and nucleotide levels.Consequently, microsatellite and chromosomal instability can be used as prognostic markers for BC, and previous experiences have shown that specific mutations are associated with a higher probability of progression and poorer survival after RC.In such a rapidly evolving tumor microenvironment (TME), genotypic and phenotypic alternations that can modulate gene and protein expression are critical for tumor invasion and the development of tumor escape mechanisms.Table 4 lists the studies addressing gene-delivery therapy for BCG-unresponsive NMIBC.A schematic summary of the gene therapies described is shown in Figure 4.  CG0070 is a replication-competent oncolytic adenovirus that targets BC cells through their defective retinoblastoma (Rb) signaling pathway, expanding the field of emerging targeted agents.Packiam et al. studied 45 BCG-responsive patients who refused RC and received intravesical CG0070 in the context of a phase II single-arm multicenter trial (NCT02365818).Overall, administration of CG0070 resulted in a CR rate of 47% at 6 months for all patients and 50% for patients with pure CIS [96].
Another potential therapeutic option for BCG-unresponsive bladder cancers is BC-819, a double-stranded plasmid with an H19 promoter and a gene encoding for diphtheria toxin-A (dtA).This plasmid is conjugated with polyethyleneimine (in vivo-jetPEI™) to enhance cell transfection.The dtA synthesis is triggered by the transcription factors of  Abbreviations are as follows: CIS: carcinoma in situ; CR: complete response; DFS: disease-free survival; GM-CSF: granulocyte macrophage-colony stimulating factor; MIBC: muscle-invasive bladder cancer; Rb: retinoblastoma; RFS: recurrence-free survival; RR: response rate; RC: radical cystectomy.
Intravesical recombinant IFNα-2b protein has been shown to be a well-tolerated molecule in BCG-unresponsive patients [101].Here, intravesical IFNα gene delivery offers a new option for the local treatment of NMIBC by significantly prolonging the duration of exposure to IFNα-2b.Nadofaragene firadenovec (rAd-IFNα/Syn3) consists of rAdIFNα, a non-replicating recombinant adenovirus vector-based gene therapy agent that delivers a copy of the human IFNα-2b gene into the urothelial cell wall [102,103], while Syn3 is a polyamide surfactant that enhances viral transduction of the urothelium [104].The in vitro reports showed that recombinant IFNα gene therapy led to local IFNα-2b production and was able to induce tumor regression [102,103].In a phase II trial, in 40 HG NMIBC patients with BCG-refractory and/or relapsing disease, 35% of the included sample were free of HG recurrence after 12 months [95].Following these encouraging results, Boorjian et al. evaluated 151 BCG-unresponsive patients within a phase III multicenter (33 US Institutions), single-arm, open-label, repeat-dose clinical study.The primary endpoint was any time CR in patients with CIS with or without an HG Ta/T1 NMIBC [94].Overall, 55/103 (53.4%)CIS patients had a CR response within 3 months of the first dose, and this response was maintained in 25/55 (45.5%) of them at 12 months [94].Nadofaragene firadenovec was well tolerated, with no dose-limiting toxicity or clinically significant treatment-related side effects, and a single dose was sufficient to achieve measurable urine IFNα [97].Overall, rAd-IFNα/Syn3 has been evaluated in four single-armed cohort trials [94,95,97,98], which found CR rates ranging from 29% to 60% (3 months) and from 29% to 35% (12 months), respectively [6].
It is worth mentioning that in 2018, the FDA granted Fast Track and Breakthrough Therapy status to rAd-IFN/Syn3, and in 2022, this therapy called ALDASTRIN received Fast Track, Breakthrough Therapy, Accelerated Approval, and Priority Review status.Therefore, it is expected to be on the market soon.
CG0070 is a replication-competent oncolytic adenovirus that targets BC cells through their defective retinoblastoma (Rb) signaling pathway, expanding the field of emerging targeted agents.Packiam et al. studied 45 BCG-responsive patients who refused RC and received intravesical CG0070 in the context of a phase II single-arm multicenter trial (NCT02365818).Overall, administration of CG0070 resulted in a CR rate of 47% at 6 months for all patients and 50% for patients with pure CIS [96].
Another potential therapeutic option for BCG-unresponsive bladder cancers is BC-819, a double-stranded plasmid with an H19 promoter and a gene encoding for diphtheria toxin-A (dtA).This plasmid is conjugated with polyethyleneimine (in vivo-jetPEI™) to enhance cell transfection.The dtA synthesis is triggered by the transcription factors of H19, an oncofetal riboregulator RNA, which is overexpressed in embryonic tissues and various human tumors.Accordingly, the diphtheria toxin is selectively expressed in cancer cells, leading to blockage of protein synthesis and cell death.Sidi et al. tested the preliminary efficacy and safety of BC-189 in 18 BCG-unresponsive patients with confirmed expression of H19.In 22% (4 out of 18) of patients, tumor markers disappeared completely without the appearance of a new tumor.Monthly maintenance therapy was given to 9 patients, 5 of whom had a disease-free survival (DFS) greater than 35 weeks [100].

Other Therapies
Photodynamic therapy (PDT), which uses an interaction between absorbed light and a retained photosensitizing agent to destroy tissue (Figure 5), has been used to treat NMIBCs after BCG treatment [105].Kulkarni et al. studied six BCG-unresponsive patients in an open-label, single-arm, dose-escalating study.The cohort was treated with photosensitizer TLD-1433-mediated PDT.Of three patients treated with the therapeutic dose, two achieved a CR after 180 days, which was confirmed at 18 months.The other patient was diagnosed with metastatic disease approximately 4 months later.PDT was well tolerated.However, all patients experienced at least one grade ≤ 2 adverse event [106].Lee and colleagues evaluated the efficacy of PDT using Radachlorin in patients with HG NMIBC who were refractory or intolerant to BCG.By including 34 patients, the authors found an RFS rate of 90.9%, 64.4%, and 60.1% at 12, 24, and 30 months, respectively.No differences were found in survival analysis for lesion size, the presence of CIS, or the number of previous BCG cycles [107].
interest as a prognostic BC biomarker [20].Hahn and the Hoosier Cancer Research Network Trial HCRN 12-157 evaluated the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non-muscle-invasive urothelial carcinoma of the bladder (NMIUC).Inclusion criteria were BCG-unresponsive NMIUC (>2 prior intravesical therapies) with increased phosphorylated FGFR3 (pFGFR3) expression evaluated centrally.The authors found an overall 6-month CR rate of 8% (33% among patients with FGFR3 mutation), whereas no response was observed in 11 mutation-negative FGFR3 patients.The reported DFS in the patient's CR was 19 months [109].Targeting the vascular endothelial growth factor (VEGF) axis was studied in NMIBC patients.VEGF is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells, and sunitinib is a VEGF receptor (VEGFR) inhibitor with anti-tumor activity against BC.Zahoor et al. studied 19 NMIBC patients who had failed primary BCG administration in a single-arm phase II study (NCT01118351).Overall, 15/19 patients completed the full course of therapy.At 12-week cystoscopy, 44% of the study group showed remission, 50% had progressive disease, and 6 had BC recurrence.Thus, treatment with sunitinib was relatively safe but not associated with improved clinical outcomes in the target population [108].
Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have gained interest as a prognostic BC biomarker [20].Hahn and the Hoosier Cancer Research Network Trial HCRN 12-157 evaluated the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non-muscle-invasive urothelial carcinoma of the bladder (NMIUC).Inclusion criteria were BCG-unresponsive NMIUC (>2 prior intravesical therapies) with increased phosphorylated FGFR3 (pFGFR3) expression evaluated centrally.The authors found an overall 6-month CR rate of 8% (33% among patients with FGFR3 mutation), whereas no response was observed in 11 mutation-negative FGFR3 patients.The reported DFS in the patient's CR was 19 months [109].
Table 5 lists the reports considering alternative treatments for BCG-unresponsive NMIBC.

Conclusions and Future Perspectives
BCG-unresponsive disease constitutes a therapeutic challenge, and the best standard of care treatment continues to be investigated.Novel combinations that improve the efficacy of pembrolizumab are currently under investigation.ICIs T-cell immunoglobulin and ITIM domain (TIGIT) and lymphocyte-activation gene 3 (LAG-3) have been shown to contribute to treatment resistance in many malignancies, and their inhibition may enhance the effect of pembrolizumab [111].In particular, TIGIT is an inhibitory receptor expressed on lymphocytes that has recently been highlighted as a major emerging target in cancer immunotherapy.By interacting with CD155 expressed on tumor cells, TIGIT can downregulate the NK functions of T cells.Thus, TIGIT has emerged as a key player in anti-tumor responses that can interfere with several steps of the cancer immunity cycle [112].On the other hand, LAG-3 holds considerable potential as it suppresses T-cell activation and cytokine secretion, thus ensuring immune homeostasis.It shows remarkable synergy with PD-1 to inhibit immune responses.Immunotherapy targeting LAG-3 is moving forward in active clinical trials, also considering the scenario of BCG-unresponsive NIMBC, and the combination of anti-LAG-3 and anti-PD-1 has shown interesting efficacy in contrasting PD-1 resistance development [113].The American Society of Clinical Oncology (ASCO) 2023 focuses on the KEYNOTE-057 (NCT02625961) cohort C, which will evaluate the efficacy and safety of the combination of pembrolizumab and vibostolimab (a TIGIT inhibitor) or favezelimab (a LAG-3 inhibitor) in patients with NIMBC.Patients will be randomly assigned in a 1:1 ratio.The primary efficacy endpoint will be the 12-month CR rate of HR NMIBC as determined by cystoscopy, cytology, biopsy, and radiologic imaging.A central pathology and radiology review will be applied [111].
In addition, encouraging results for erdafitinib, an oral selective pan-FGFR tyrosine kinase inhibitor, were recently highlighted by the THOR-2 study (NCT04172675) in patients with BCG-unresponsive CIS with FGFRalt with or without the papillary disease [114].
Tislelizumab (BGB-A317) is a humanized IgG4 anti-PD-1 monoclonal antibody designed to minimize binding to FcγR on macrophages.In preclinical studies, binding to FcγR on macrophages has been shown to compromise the anti-tumor activity of PD-1 antibodies through the activation of antibody-dependent macrophage-mediated killing of T effector cells.Previously, tislelizumab demonstrated clinical advantages in patients with locally advanced or metastatic PD-L1-positive UC [115].Li et al. evaluated the efficacy and safety of tislelizumab plus radiotherapy as a bladder-sparing strategy in 14 BCG-unresponsive patients and found promising results with a bladder-preservation rate of 100% at 24 months and a remarkable OS rate at the same time point [116].
Enfortumab vedotin (EV) is an antibody-drug conjugate directed against Nectin-4.In EV-301, Powles et al. found an OS benefit for EV in patients with locally advanced or metastatic UC who had previously received platinum-based therapy and a PD-1 or PD-L1 inhibitor [117].Kamat et al. presented EV-104 (NCT05014139) at ASCO 2023.This trial, a phase 1, open-label, multicenter, dose-escalation, and dose-expansion study, will evaluate the safety, tolerability, and anti-tumor activity of intravesical EV in BCG-unresponsive NMIBCs [118].The study is currently enrolling in the US, with additional recruiting sites in Canada and Europe.
Many agents in single or combined administration have been evaluated in this scenario, obtaining disparate response rates.Starting from conventional chemotherapy up to immunotherapy and new targeted therapies, it seems plausible that the biological aggressiveness behind the BCG-unresponsive disease may harbor various vulnerabilities that can be targeted at different stages with different compounds, mirroring a remarkable interpatient and intratumor heterogenicity.As new therapeutic options emerge, selective biomarkers for treatment selection and clinical trial design are urgently needed.In addition, the optimal sequence of treatments remains to be elucidated.Therefore, novel therapeutics are currently being developed for patients with BC recurrence or persistence after BCG treatment.For example, Taizhou Hanzhong Biomedical Co., Ltd.(Taizhou, China) is supporting a single-arm, open-label, multicenter study to evaluate the efficacy and safety of HX008 in patients with BCG-unresponsive non-muscle-invasive bladder cancer [119] Similar to pembrolizumab, HX008 is a humanized anti-PD-1 monoclonal antibody that inhibits PD-1-PD-L1 binding, leading to T-cell activation [120].This clinical trial started in September 2020, is in phase II, and is expected to be completed in December 2023.
Instead, enGene Inc. (Vancouver, BC, Canada) is sponsoring an open-label, multicenter study to evaluate the efficacy of intravesical administration of EG-70 in patients with BCG-unresponsive non-muscle-invasive bladder cancer [121].EG-70 is a non-viral vector plasmid that encodes IL-12 and RIG1 to trigger, respectively, the adaptive and innate immune responses.The intravesical administration ensures a localized immune response by delivering the plasmids into the bladder mucosal tissue.The clinical trial was started in April 2021 and is expected to provide complete data in February 2026.
In conclusion, BCG continues to play a central role in the treatment of high-risk NMIBC.This immunotherapy is highly effective in preventing the recurrence and progression of NMIBC and remains the treatment of choice today.However, there is a significant proportion of patients who are classified as BCG-unresponsive.The aim of our review was to examine the role of these alternative therapies, focusing on the new molecular targets.
In addition to BCG, there are other intravesical therapies used in the treatment of NMIBC.Gemcitabine, docetaxel, valrubicin, and other therapies have shown a potential role in the treatment of urothelial carcinoma.All of these therapies aim to prevent tumor recurrence, treat high-risk or refractory NMIBC, or treat certain patients who cannot tolerate or do not respond to BCG.Our review highlights the role and limitations of these various drugs in the treatment of bladder cancer.
Hyperthermia has been studied as a potential treatment option for NMIBC.In combination with other therapies such as intravesical chemotherapy, hyperthermia has shown promise in improving treatment outcomes in NMIBC patients, particularly those with high-risk or recurrent tumors.Although hyperthermia has shown promising results in clinical trials, it is important to note that it is not yet considered the standard first-line treatment for NMIBC.Research is ongoing to determine the optimal protocols, safety, and long-term benefits of hyperthermia in combination with other treatments for NMIBC.
The use of immunotherapy and anti-inflammatory agents in NMIBC is an area of active research and ongoing clinical trials.Although some of these agents have shown promising results, further demonstration of their safety and efficacy in the treatment of NMIBC is needed.Advances in the field of immunotherapy hold great potential to transform the treatment of NMIBC and improve outcomes for patients with this type of bladder cancer.However, it is critical to recognize that not all patients will benefit from these therapies, and the selection of the most appropriate treatment should be guided by individual patient characteristics and clinical considerations.
Gene therapy for the treatment of NMIBC, while very promising, is still in the early stages of development, and further research is needed to optimize its efficacy and safety.Challenges in gene delivery, immune response regulation, and targeted gene expression need to be addressed.Clinical trials are currently underway to evaluate the safety and efficacy of gene therapy for NMIBC.Future advances in this field may provide new treatment options for patients with this type of bladder cancer.

Figure 1 .
Figure 1.Active molecules and mechanism of action of intravesical chemotherapy.The image was created with BioRender.com.

Figure 1 .
Figure 1.Active molecules and mechanism of action of intravesical chemotherapy.The image was created with BioRender.com.

Figure 2 .
Figure 2. Scheme and mechanism of chemo-hyperthermia treatment.The image was create BioRender.com.

Figure 2 .
Figure 2. Scheme and mechanism of chemo-hyperthermia treatment.The image was created with BioRender.com.

Table 1 .
Studies on intravesical administration of chemotherapy in NMIBC patients after BCG.

Table 2 .
Chemo-hyperthermia and MMC administration in NMIBC patients following BCG.

Table 3 .
Immunotherapy and inflammation-targeted agents' administration in NMIBC patients following BCG.

Table 4 .
Gene-delivered agents' administration in NMIBC patients following BCG.

Table 5 .
Other agents' administration in NMIBC patients following BCG.