EGFR (epidermal growth factor receptor), a member of the ERBB/HER protein family, features intracellular, transmembrane, and signal-transduction domains with tyrosine kinase. The activated EGFR protein stimulates a series of downstream pathways, including the RAS/RAF/MAPK pathway and the PI3K/AKT/mTOR pathway which mainly promote cell proliferation and restrict apoptosis. However, functional EGFR mutations or overexpression of the EGFR protein will result in excessive cell proliferation which will eventually induce malignant transformation.

Lee et al. (2017) reported EGFR amplification in 23.5% of patients (4/17) by detecting somatic copy number aberrations. More recently, Varadi et al. (2023) described EGFR mutations in 9.1% (3/33) of UrC cases by applying next-generation sequencing. These authors detected two samples with amplification and one sample with a missense mutation. Both missense mutations and amplification can result in a functional EGFR protein. However, other studies failed to identify EGFR mutations in patients with UrC (Jordan et al., 2015; Lee et al., 2016; Modos et al., 2016; Modos et al., 2017). Statistical analysis has shown that the estimated occurrence of EGFR mutations was 14.1%–38.4% in patients with non-small cell lung cancer (Zhang et al., 2016). In contrast, EGFR aberrations only presented in 6.1% (8/132) of UrC cases while the meta-analytic pooled occurrence of EGFR mutations was 14% (95% CI: 10%–20%).

Thus far, two distinct therapeutic strategies have been developed to resist the pathogenic effects of mutation-activated EGFR signaling, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Furthermore, numerous EGFR targeted agents have been developed and approved for various types of cancers (Abourehab et al., 2021). Although no anti-EGFR antibodies and TKIs have been approved for the treatment of advanced UrC, some studies have attempted to explore the potential benefits of this novel treatment for UrC. In a recent study, one patient with metastatic disease who harbored EGFR amplification and wild-type KRAS exhibited a partial response for more than 8 months after receiving treatment with cetuximab, an anti-EGFR monoclonal antibody (Collazo-Lorduy et al., 2016). A previous phase I study evaluated the efficacy of the EGFR-TK inhibitor Iressa for various advanced tumors, including one UrC patient featuring the overexpression of EGFR; this case showed a significant reduction in tumor size by 55% following Iressa treatment (Goss et al., 2005). In addition, a patient with recurrent UrC achieved stable disease and improvement of symptoms for 5 months after receiving sunitinib, a tyrosine kinase inhibitor (Testa et al., 2014).

These promising data demonstrate that anti-EGFR-TKI therapy might also be effective for UrC. However, numerous mutations of these downstream pathways, including KRAS, BRAF, PIK3CA, PTEN, and MET gene mutations could increase resistance against anti-EGFR treatment (Dietel et al., 2015). Therefore, we hypothesized that the identification of actionable gene alterations in these downstream pathways is warranted before adopting EGFR targeted treatment for UrC (Wang et al., 2010; Dietel et al., 2015).

RAS proteins, a small family of GTPase binding GTP/GDP proteins, regulate normal growth, proliferation, and differentiation in normal cells. KRAS (Kirsten rat sarcoma viral oncogene homolog), NRAS (neuroblastoma rat sarcoma viral oncogene homolog) and HRAS (Harvey rat sarcoma viral oncogene homolog) proteins are all encoded by the RAS gene family. When RAS aberrations occur, the RAS signaling pathway remains continuously activated without upstream stimulation of the EGFR receptor, thus resulting in abnormal cell growth, proliferation, and differentiation; this eventually results in malignant transformation. KRAS mutation is the most frequently altered form of the three RAS genes, accounting for 86% of RAS mutant cancer cases. Lee et al. (2016) reported KRAS mutations in 2 out of 17 patients based on whole exome sequencing and application of the Onco Scan platform while Cornejo et al. (2016) used the next-generation sequencing approach and identified KRAS mutations in 50% (8/16) of UrC samples; one case featured a mutation of the NRAS gene. More recently, Reis et al. (2018) performed targeted next-generation sequencing of 70 urachal adenocarcinomas and showed that 14 out of 70 samples (21%) had KRAS missense mutations. In addition, Nagy et al. (2020) reported KRAS mutations in 8 out of 26 (35%) samples by using the ionTorrent technology.

In the present analyses of UrC patients, we identified the presence of KRAS mutations in 28.3% of cases; in a previous study, KRAS missense mutations were found in 40% of CRC cases (Shitara et al., 2020); in addition, the meta-analytic pooled occurrence of KRAS mutations was 39% (95% confidence interval [CI]: 35%–50%). Sirintrapun et al. (2014) reported KRAS mutations in codon 12 (p.G12D, p. G12C, p. G12S, and pG12V) in some cases of UrC while Modos et al. (2016) reported that 6 out of 22 (27%) cases presented with KRAS alterations; 3 out of 6 KRAS mutations occurred in codons 61 and 146. In addition, Collazo et al. (Collazo-Lorduy et al., 2016) used a targeted exome sequencing approach and reported KRAS mutation at G13D and G12v in 9 UrC samples. Singh et al. (2016) also identified a G13D KRAS mutation in one patient with UrC. In another study, Cha et al. (Dienstmann et al., 2020) performed whole exome sequencing in adolescent and young adults with metastatic cancers; one UrC patient was identified to possess a G13D KRAS mutation. Using Sanger sequencing, Hang et al. (Hang and Pan, 2017) identified KRAS mutations in 5 out of 12 cases (p.G12V, 2 cases:p.G13D, 2 cases; p.G12R, 1 case).

Notably, in CRC, the most common KRAS mutation is a somatic missense mutation in codon 12 which leads to a single amino acid substitution (Zhu et al., 2021). In this review, we identified a missense mutation in codon 12 (Sirintrapun et al., 2014; Loh et al., 2016b; Collazo-Lorduy et al., 2016; Modos et al., 2016; Hang and Pan, 2017; Riva et al., 2019; Varadi et al., 2023) and codon 13 (Collazo-Lorduy et al., 2016; Singh et al., 2016; Hang and Pan, 2017; Dienstmann et al., 2020) in 23% (23/100) and 5% of UrC samples, while KRAS missense mutations in codon 61 (Modos et al., 2016; Riva et al., 2019) and codon 146 (Modos et al., 2016) were present in 2% and 2% of cases, respectively. Collectively, these data revealed that the most common mutation of the KRAS gene was the missense mutation in codon 12; this is similar to the KRAS mutational pattern in CRC (Cha et al., 2016).

Activated RAS mutations interrupt upstream transduction signals; this might result in a poor response to anti-EGFR therapy or double HER2 blockade in patients with EGFR or ERBB2 amplifications (Cancer Genome Atlas Network, 2012; Siravegna et al., 2019). Given the similarity in KRAS mutational patterns between CRC and UrC, it is possible that the KRAS mutational status might also restrict targeted treatment options for this rare tumor.

Although no previous study has reported the application of KRAS inhibitors for patients with UrC, several agents targeting mutant RAS protein are available and deserve our attention. Sotorasib and adagrasib, specific and irreversible KRAS (G12C) protein inhibitors, have shown modest anti-tumor effects and tolerable toxicity in the treatment of advanced solid tumors with KRAS (G12C) mutation, including colorectal cancer (Dienstmann and Tabernero, 2016), lung cancer (Fakih et al., 2022), and pancreatic cancer (Sabari et al., 2022). A combination of these two agents with other targeted agents is under evaluation to increase potential efficacy and reduce potential drug resistance (NCT05074810, NCT04185883, and NCT03785249). In addition, several clinical trials of BI-1701963, a non-specific inhibitor of KRAS for KRAS-mutant tumors are ongoing; these trials might provide new insights for the treatment of UrC patients with KRAS mutation.

The BRAF proto-oncogene, a member of the RAF gene family, acts as a significant mediator in the RAS/RAF/MAPK pathway in which the BRAF protein mediates signal transduction between RAS and MAPK kinase (MAPKK/MEK1/2). Alterations in the BRAF gene result in the continuous activation of downstream pathways without upstream signals and therefore promotes cell proliferation and malignant transformation.

Statistical analysis has demonstrated that 30%–60% of melanoma cases harbor BRAF mutations; however, in our present study, only 6.7% (12/180) of UrC patients possessed BRAF mutations. In accordance with RAS mutation, many studies have shown that patients possessing BRAF mutations also exhibit drug resistance to cetuximab or panitumumab (anti-EGFR monoclonal antibodies) in CRC, thus suggesting the potential role of these drugs in anti-EGFR therapy for patients with UrC. Although BRAF-targeted agents such as vemurafenib, dabrafenib, and encorafenib, have been approved for the management of melanoma, the efficacy of BRAF inhibitors for UrC remains unclear. In our study, only four studies reported MAP2K1 mutations in of 8.6% of 58 UrC cases, Notably, single-agent therapy involving trametinib, a MEK inhibitor, resulted in stable disease for 10 months in one patient with metastatic UrC and MAP2K1 mutation (Loh et al., 2016a). This encouraging finding demonstrated that patients bearing mutations in the MAPK pathway particularly MAP2K1 mutations) might acquire potential efficacy from MEK inhibitors. More clinical investigations are needed to validate the efficacy of MEK inhibitor for specific UrC patients.

The phosphoinositide 3-kinase (PI3K) pathway is one of the downstream pathways of the ERBB/HER protein family and is activated by various oncogenes and growth factor receptors [such as EGFR and human EGFR 2 (HER2)]. Furthermore, aberrantly activated PI3K signaling activity is considered as one of the characteristics of tumorigenesis. PI3CA encodes p11oalpha, a catalytic subunit of PI3K that regulates the PI3K/AKT/mTOR pathway and promotes cell survival and proliferation. PIK3CA abnormalities could result in the abnormal activation of the PI3K pathway.

In the present analyses, we found that the prevalence of PIK3CA mutations in UrC was 5.6% (12/215). Notably, activating PIK3CA alterations was related to a negative response to anti-EGFR-TKI therapy in CRC (Day et al., 2013; Strickler et al., 2023), thus suggesting that the status of PIK3CA genotype may be an important factor when selecting the choice of anti-EGFR therapy for patients with UrC.

Generally, the PI3K/AKT/mTOR pathway has two major targets: PI3K and mTOR. At present, several PI3K inhibitors such as idelalisib, duvelisib, and copanlisib, have been successfully marketed for the treatment of PIK3CA-mutant head and neck tumors, breast cancer or lymphoma. mTOR inhibitors, such as everolimus, are mainly used to treat advanced renal cell carcinoma. Indeed, there are no previous publications relating to the treatment of UrC with mTOR inhibitors or PI3K inhibitors.

Breast cancer susceptibility gene 1/2 (BRCA1/2), a tumor suppressor gene, participates in the homologous recombination repair process, and may help to safeguard genomic integrity via precise DNA repair. Furthermore, the inactivation of BRCA1/2 genes has been observed in many forms of cancer, particularly in breast and ovarian cancer. Due to the synthetic lethality of poly (ADP-ribose) polymerase (PARP) inhibition in the presence of BRCA1/2 mutations, tumor cells that possess BRCA1 and BRCA2-deficiency are exquisitely sensitive to treatment with PARP inhibitors (Cathomas, 2014). Furthermore, PARP inhibitors have recently gained FDA approval for patients with metastatic breast cancer and germline BRCA mutation (Bryant et al., 2005).

In this review, we found that only one study detected BRCA abnormalities in UrC patients with a prevalence of 12.1% (4/33). Notably, some institutions have investigated the efficacy of agents targeted to the BRCA gene. Seto et al. (2019) reported the successful use of rucaparib, a PARP inhibitor, in a patient with metastatic UrC who possessed a germline BRCA1 mutation, thus suggesting that patients with BRCA mutations might be candidates for the use of PARP inhibitors. In a phase I dose escalation study, a UrC patient receiving niraparib was reported to experience disease progression during therapy; however, the BRCA status of patients with UrC was not clarified (Yonemori et al., 2021). Collectively, these data indicated that PARP inhibitors could represent a therapeutic option for UrC patients possessing BRCA deficiency.

The expression of the PD-L1 on tumor cells has validated and significant efficacy for PD-1 inhibition in cases of non-small-cell lung cancer (Mok et al., 2019). However, the predictive value of PD-L1 expression for the treatment of UrC with PD1 inhibitors is currently unknown, although some institutions have shared their experience with regards to PD-L1 expression profile. For example, Reis et al. (2018) performed PD-L1 analyses using clone 22C3; in 10 out of 63 cases (15.9%), a specific level of PD-L1 expression was detected in tumor cells. Positive PD-L1 expression was associated with a shorter PFS (p = 0.002). In another study, Maurer et al. (2020) evaluated PD-LI expression in glandular bladder tumors and stained all available samples (including samples from three UrC patients) with four different anti-PD-L1 antibody clones (28–8, SP142, SP263, and 22C3); none of the three UrC patients tested were positive for PD-L1 expressing immune cells and tumor cells.

In another study, Zhang et al. (2022) used immunohistochemistry to investigate the characteristics of the immune microenvironment in 37 UrC patients. These authors found that the proportion of immune cells that were positive for PD-L1 protein expression was 35.14% (13/37). Only one case (2.78%) was found to possess tumor cells that were positive for PD-L1 membranous expression. In addition, immune cells that were positive for PD-L1 expression were only marginally associated with a short overall survival (OS) (p = 0.3700) and disease free survival (DFS) (p = 0.5400) in patients with UrC. Notably, no significant correlation was identified between the protein expression of PD-L1 in immune cells, histological type, and tumor stage, After summarizing all available data from 103 UrC patients with PD-L1 expression levels, 11 (10.7%) were positive for PD-L1 expression. However, 35.14% (13/37) of cases expressed PD-L1 on tumor-associated immune cells,

Mismatch repair pathways serve as pivotal regulators in identifying and repairing mismatched nucleotides during DNA replication or genetic recombination and therefore guarantees genomic integrity and stability. The MMR system consists of a series of DNA mismatch repair proteins, including MLH1, PMS2, MSH2, and MSH6; these can be identified by immunohistochemical approaches in clinical practice. A deficiency of MMR (dMMR) proteins may lead to spontaneous hypermutation alterations and therefore result in microsatellite instability (MSI) (Zhao et al., 2019). Furthermore, research has revealed high consistency between dMMR and MSI (Cicek et al., 2011). Furthermore, dMMR status has been reported in approximately 15% of CRC cases (Zhao et al., 2019) whereas the loss of MMR protein was detected in 10% of 210 UrC samples. In another study, Sirintrapun et al. (2014) described one case involving the loss of MSH2, MSH6 and PMS2 proteins. In another study, Kardos et al. (2017) used a targeted exon sequencing method to identify the presence of dMMR in 25% (3/12) of urachal tumors, including two cases with MSH6 loss and one case with the loss of MSH2 and polymerase epsilon complex (POLE). More recently, Zhang et al. (2022) found that 8.1% (3/37) of UrC patients suffered from dMMR, as determined by IHC staining. However, Hang et al. (Hang and Pan, 2017) also used immunohistochemistry and demonstrated that MMR proteins (MLH1, MSH2, MSH6 and PMS2) were preserved in all UrC samples tested. Similarly, Reis et al. (2018) investigated 56 cases by performing MSI analyses and identified only one case that was negative for MSH2 expression. Notably, patients with MMR defects exhibited a considerable response to PD-1 blockade regardless of tumor type (Dudley et al., 2016) Pembrolizumab, an immune checkpoint inhibitor, was approved in 2017 for the treatment of MMR defects in solid tumors (Marcus et al., 2019). Similarly, Kardos et al. (2017) reported one UrC patient harboring a MSH6 mutation who received atezolizumab, an anti-PD-L1 antibody. This patient benefited from the treatment, thus suggesting that dMMR status may represent a favorable biomarker for immunotherapy in UrC.