Abbreviations are as follows: BCG: Bacillus Calmette–Guerin; CFS: cystectomy-free survival CIS: carcinoma in situ; CR: complete response; CRR: complete response rate; CSM: cancer-specific mortality; CSS: cancer-specific survival; Gem/Doce: gemcitabine/docetaxel; HG: high grade; IFN: interferon; MIBC: muscle-invasive bladder cancer; MMC: mitomycin C; Nab: nanoparticle albumin-bound; NMIBC: non-muscle-invasive bladder cancer; NR: non-responders; OS: overall survival; PFS: progression-free survival; PR: partial response; Pts: patients; RC: radical cystectomy; RFS: recurrence-free survival; TNF: tumor necrosis factor; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. In this scenario, among the chemotherapeutic agents studied, gemcitabine, a deoxycytidine nucleoside analog that can inhibit DNA synthesis and is a cornerstone in the systemic treatment of MIBC in both neoadjuvant and adjuvant settings, may be the one that receives more attention [24]. Gemcitabine has been administered as a single agent by some authors ^{[25][37][38][39][40][41][42][43][44][45][46][47][48][49][51][52][53]}[25,37,38,39,40,41,42,43,44,45,46,47,48,49,51,52,53], while other series have tested combinations with various other drugs such as docetaxel ^{[25][27][29][33]}[25,27,29,33], cabazitaxel and cisplatin [28], oral everolimus [30], and mitomycin C (MMC) ^[32][34][32,34]. Gemcitabine administration regimens are quite heterogeneous in the published literature, with some studies suggesting an induction regimen only ^{[38][47][48][51][53]}[38,47,48,51,53] and others recommending a maintenance regimen in responders ^{[26][37][42][43][45][52]}[26,37,42,43,45,52]: no clear and standardized regimen can be found in either single-agent or drug combination studies. 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 researchtudy, 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 researchtudy, 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][31,40,41], valrubicin, and doxorubicin ^[33][46][33,46] have been studied with conflicting results. Docetaxel alone resulted in a CR in 56% to 59% of patients ^[36][40][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.

3. 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].

4. 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][57][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 ^[58][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 ^[59][68] and intravenous routes and as a single agent ^[58][67].

5. Gene Therapy

Genetic instability is an essential early step in the development of BC ^[60][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. A schematic summary of the gene therapies described is shown in Figure 2. Intravesical recombinant IFNα-2b protein has been shown to be a well-tolerated molecule in BCG-unresponsive patients ^[61][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 ^[62][63][102,103], while Syn3 is a polyamide surfactant that enhances viral transduction of the urothelium ^[64][104]. The in vitro reports showed that recombinant IFNα gene therapy led to local IFNα-2b production and was able to induce tumor regression ^[62][63][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 ^[65][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 ^[66][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 ^[66][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 ^[67][97]. Overall, rAd-IFNα/Syn3 has been evaluated in four single-armed cohort trials ^{[65][66][67][68]}[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 ^[69][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 ^[70][100].