FGFR3 has been found to be mutated in up to 64% of cases of bladder cancer; FGFR3 mutations tend to be exclusive of RAS mutations ,TP53 overexpression, TP53 mutation, but not PIK3CA mutations. However, subsets of cases with co-mutations have been described. FGFR3 mutations (including Y373C) are believed to lead to constitutive activation of the receptor and activation of the RAS-MAPK pathway. FGFR3 mutations are often seen in non-muscle invasive bladder cancers and tend to correlate with low stage and grade; however FGFR3 mutations have also been described in muscle-invasive bladder cancer. Targeted therapies with FGFR3 inhibitors have been explored in patients with bladder cancer.

When mutated, HRAS can act as an oncogene. Somatic HRAS mutations have been associated with some cases of upper and lower urinary tract urothelial carcinoma, thyroid, and kidney cancers and in nevi. The frequency of HRAS mutations in upper and lower urinary tract urothelial carcinoma are about 10%. Interestingly, it has been shown that the HRAS mutation in lower urinary tract urothelial carcinoma may occur in tumors without FGFR3 mutations.

NRAS is a member of the RAS family of oncogenes and activating mutations of NRAS have been reported in a wide variety of tumors including occasional cases of bladder cancer. This finding may influence targeted therapy options.

PIK3CA mutations activate the PI3K-PTEN-AKT pathway which is downstream from both the EGFR and the RAS-RAF-MAPK pathways.The somatic mutations found thus far in PIK3CA are oncogenic, and the majority of them are clustered within exon 9 and 20 (helical and kinase domains). Activating mutations in PIK3CA are found are found in a wide variety of human cancers including 27% urothelial bladder cancers, with a higher prevalence in low grade tumors. Up to 13.6% of renal pelvic urothelial carcinomas also harbor activating somatic mutations in PIK3CA gene. The role of PIK3CA mutations as prognosticators of outcome or predictors of therapeutic response awaits further evaluation.

Activating extracellular domain ERBB2 mutations (S310F, S310Y, R157W) have been identified in urothelial carcinoma (enriched in micropapillary variant) and adenocarcinomas of breast and lung. These activating mutations may have therapeutic potential in some clinical settings.

MDM2 encodes an E3 ubiquitin ligase that regulates tumor suppressor protein (eg, TP53) turnover through proteasomal degradation. MDM2 overexpression or amplification has been detected in a variety of different cancers including a subset of bladder cancer. Small molecular inhibitors of the MDM2:p53 axis are currently in early phase clinical trials for a number of malignancies.

EGFR has been reported to show increased expression in a subset of bladder cancers and may be a targetable alteration in some clinical settings.

KRAS is a gene that encodes one of the several proteins in the growth factor signaling pathway(s) and is important in the development and progression of a variety of cancers. KRAS can harbor oncogenic mutations that yield a constitutively active protein. The frequency of the KRAS gene mutations in urothelial carcinoma of urinary bladder is very low (3% to 7%), and these mutations occur in all stages and grades. In the context of bladder tumors, mutations in the KRAS gene do not appear to be predictors for recurrence-free, progression-free and disease-specific survival according to some studies.

Germ line mutations of mutations in either TSC1 or TSC2 are found in 75-90% of cases of tuberous sclerosis complex (TSC), an autosomal dominant tumor syndrome associated with variable clinical phenotype including several hamrtomas and benign tumors. In addition, somatic alterations in these genes may occur in some tumor types. TSC1 and TSC2 both are tumor suppressor genes and their inactivation occurs by a classical two-hit mechanism. TSC1 is located on chromosome 9q34 and encodes hamartin. TSC2 is located on chromosome 16p13 and encodes tuberin. Hamartin and tuberin interact with and regulate a variety of proteins. These are negative regulators of the mTOR pathway, which is important for cell proliferation and frequently found activated in tumors. Mutation or deletion of TSC1 or TSC2 is found in 9-16 % of urothelial bladder tumors and up to 3% of clear cell renal cell carcinomas. More than 50% of bladder tumors of all grades and stages show LOH for markers on chromosome 9 and the TSC1 locus at 9q34 is a common critical region of deletion. Therefore, mTOR inhibitors have been identified as potential therapies for TSC1-mutated bladder cancers in some studies. LOH for the TSC1 or TSC2 locus has been described in 22% of 86 human lung cancer specimens. However, TSC1/2 sequence alterations are infrequent in lung and other epithelial malignancies.

Germ line mutations of mutations in either TSC1 or TSC2 are found in 75-90% of cases of tuberous sclerosis complex (TSC), an autosomal dominant tumor syndrome associated with variable clinical phenotype including several hamrtomas and benign tumors. In addition, somatic alterations in these genes may occur in some tumor types. TSC1 and TSC2 both are tumor suppressor genes and their inactivation occurs by a classical two-hit mechanism. TSC1 is located on chromosome 9q34 and encodes hamartin. TSC2 is located on chromosome 16p13 and encodes tuberin. Hamartin and tuberin interact with and regulate a variety of proteins. These are negative regulators of the mTOR pathway, which is important for cell proliferation and frequently found activated in tumors. Mutation or deletion of TSC1 or TSC2 is found in 9-16 % of urothelial bladder tumors and up to 3% of clear cell renal cell carcinomas. More than 50% of bladder tumors of all grades and stages show LOH for markers on chromosome 9 and the TSC1 locus at 9q34 is a common critical region of deletion. Therefore, mTOR inhibitors have been identified as potential therapies for TSC1-mutated bladder cancers in some studies. LOH for the TSC1 or TSC2 locus has been described in 22% of 86 human lung cancer specimens. However, TSC1/2 sequence alterations are infrequent in lung and other epithelial malignancies.

FGFR3 mutations may be associated with response to the multi-targeted tyrosine kinase inhibitor pazopanib.

Although FGFR2 mutation in a patient with oral squamous cell carcinoma was reported to be associated with response to the multitargeted tyrosine kinase inhibitor pazopanib, FGFR2 mutations in bladder cancer are very rare. The clinical significance of this finding is unknown.

The CDKN2B gene locus is altered in up to approximately 60% of bladder cancer, most commonly as a homozygous deletion, and frequently with concurrent deletion of the CDKN2A locus. CDKN2A/CDKN2B loss may be associated with increased sensitivity to CDK4/6 inhibitors. The efficacy and toxicity profiles of these inhibitors in the context of a variety of cancer types are currently under evaluation in clinical trials.

Some mutations in BRCA2 may be associated with sensitivity to PARP inhibitors. The effect of this missense mutation is not entirely clear. Drug: Rucaparib Niraparib Olaparib

In lung adenocarcinomas, CRKL amplification may be associated with resistance to EGFR-directed therapy (Cheung et al, 2011). However, its significance in urothelial cancer is yet to be determined.

This gene encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Amplification of ERBB3 and/or overexpression of its protein have been reported in numerous cancers, including prostate, bladder, non small cell lung cancer, endometrial cancer and breast tumors. Several ERBB3 inhibitors are under various clinical trials against different types of solid tumors, including non small cell lung cancer, breast cancer, ovarian cancer and gastric cancer.

The protein of CCND1 (Cyclin D 1) belongs to the highly conserved cyclin family, functioning as regulators of CDK kinases. This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This protein has been shown to interact with tumor suppressor protein Rb and the expression of this gene is regulated positively by Rb. Amplification of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors. Cyclin D1 and the mechanisms it regulates have the potential to be a therapeutic target for cancer drugs, including inhibition of Cyclin D1, induction of Cyclin D1 degradation, and inhibition of Cyclin D1/CDK 4/6 complex.

The protein of CCND1 (Cyclin D 1) belongs to the highly conserved cyclin family, functioning as regulators of CDK kinases. This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This protein has been shown to interact with tumor suppressor protein Rb and the expression of this gene is regulated positively by Rb. Amplification of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors. Cyclin D1 and the mechanisms it regulates have the potential to be a therapeutic target for cancer drugs, including inhibition of Cyclin D1, induction of Cyclin D1 degradation, and inhibition of Cyclin D1/CDK 4/6 complex.

Activation of FGFR protein family can lead to the activation of RAS-MAPK and PI3K-AKT pathways. Amplification of FGFR2 has been observed in lung adenocarcinoma, lung squamous cell carcinoma, endometrial carcinoma, urothelial carcinoma, germ cell tumor and breast cancers. Anti-FGFR2 agents are actively under multiple clinical trials against many types of solid tumor, including lung squamous cell carcinoma, gastric cancer, endometrial cancer, and cholangiocarcinoma. Germeline mutations in FGFR2 are also associated with multiple craniosynostosis syndromes.

SMAD4 is a tumor suppressor gene encoding an intracellular mediator in the transforming growth factor β (TGF β) signal transduction pathway. SMAD4 mutations have been reported in up to 2% of urothelial carcinomas. The predictive and prognostic significance of SMAD4 mutations in urothelial carcinoma needs further elucidation. Results should be interpreted in conjunction with other laboratory and clinical findings.

This mutation is located at extracellular domain. Patients with mutation of ERBB2 S310F, which is very close to S295 and also an extracellular domain, have been treated successfully with anti-HER agents (Jia et al, 2014; Vornicova et al, 2014). Although mutation at extracellular domain does not elevate HER2 protein level (negative for IHC), it might still activate HER2 protein or it’s downstream signaling.

AKT1 mutations have been reported in a variety of tumor types such as endometrial, lung, breast, colorectal, ovarian, urothelial and prostate cancers. The mutations are mutually exclusive from PIK3CA mutations. Increased expression and activation of AKT1 observed in many cancers is caused by a variety of different mechanisms including genomic alterations of AKT1, PIK3CA, PTEN, RAS family members, or growth factor receptors. Gain-of-function alterations of AKT1 can lead to neoplastic transformation in model systems, and is a potential target for therapeutic strategies. The E17K variant is by far the most frequent AKT1 mutation reported, implicating it as an important tumor promoting event.

This gene is a known cancer gene. ARID1A/BAF250A subunit of the SWI/SNF (BAF) chromatin remodeling complex has emerged as recurrently mutated in a broad array of tumor types and a potential tumor suppressor. There is evidence indicating that ARID1A-mutated cancers may be subjected to therapeutic intervention.

Somatic mutations in TP53 are frequent in human cancer. Germline TP53 mutations cause of Li-Fraumeni syndrome, which is associated with a range of early-onset cancers. The types and positions of TP53 mutations are diverse. TP53 mutations may be potential prognostic and predictive markers in some tumor types, as well as targets for pharmacological intervention in some clinical settings. The IARC TP53 Database (http://www-p53.iarc.fr/) is a useful resource which catalogues TP53 mutations found in cancer.

BRAF is a member of the RAF-family of kinases which plays an important role in the RAS-RAF-MEK-ERK mitotic signaling pathway. BRAF V600E lies within the activation segment of the kinase domain of the BRAF protein and confers a gain of function. BRAF mutations are infrequent in urothelial carcinoma and are identified in 3-5% of cases. Various BRAF inhibitors (Vemurafenib, Dabrafenib) have been FDA approved for therapy for some tumor types in certain settings. The use of BRAF inhibitors in a number of other cancer types harboring BRAF V600E mutations are under investigation (clinicaltrials.gov). The clinicopathologic effects of BRAF in urothelial carcinoma remains to be fully elucidated.

CDKN2A gene functions as an important tumor suppressor via induction of cell growth arrest and senescence. Majority of the CDKN2A mutations result in loss or decreased binding to CDK4/6 leading to uncontrolled cell growth through inactivation of RB and p53 pathways. CDKN2A alterations have been reported as frequent mutations in squamous cell carcinomas of different primary sites including the lung, head and neck, and skin. CDKN2A R58* results in a premature truncation and confers a loss of function to the CDKN2A protein as demonstrated by loss of CDK binding. CDKN2A/CDKN2B loss may be associated with increased sensitivity to CDK4/6 inhibitors. The efficacy and toxicity profiles of these inhibitors in the context of a variety of cancer types are currently under evaluation in clinical trials. The clinical significance of the loss of one copy of CDKN2A remains to be fully elucidated in squamous cell carcinoma of the bladder. These results should be interpreted in the clinicopathologic context.

Balversa (erdafitinib) has been FDA approved for treatment of urothelial carcinoma with susceptible FGFR3 or FGFR2 genetic alterations. FGFR3 is a receptor tyrosine kinase in the RAS-MAPK and PI3K-AKT pathways. FGFR3 has been found to be mutated in up to 64% of cases of bladder cancer and 40% of upper urothelial tract (ureter and renal pelvis) urothelial carcinomas. FGFR3 mutations tend to be exclusive of RAS mutations ,TP53 overexpression, TP53 mutation, but not PIK3CA mutations. However, subsets of cases with co-mutations have been described. Gain of function FGFR3 mutations (including FGFR3 R248C and FGFR3 S249C) are believed to lead to constitutive activation of the receptor and activation of the RAS-MAPK pathway. FGFR3 mutations are often seen in non-muscle invasive bladder cancers and tend to correlate with low stage and grade; however, FGFR3 mutations have also been described in muscle-invasive bladder cancer.

SMARCB1 is a member of the SWI/SNF chromatin remodeling complex and regulates transcription of several genes involved in cell proliferation. SMARCB1 mutations are rare in urothelial carcinomas and are found in about 2% of cases. SMARCB1 A203T is considered a variant of uncertain significance and has not been well classified in the literature. This variant has been reported a somatic mutation in a case of colon adenocarcinoma however this variant has also been reported a germline finding in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/25553173/). These results should be interpreted in the clinicopathologic context.

BRAF is a member of the RAF-family of kinases which plays an important role in the RAS-RAF-MEK-ERK mitotic signaling pathway. The K601E mutation results in an amino acid substitution at position 601 in BRAF, occurs within the highly conserved motif of the kinase domain. Most mutant BRAF proteins, such as K601E, have increased kinase activity and are transforming in vitro. BRAF mutations are infrequent in urothelial carcinoma and are identified in 3-5% of cases. Preclinical studies are investigating the use of MEK and RAS inhibitors in BRAF K601E mutant cell lines. The clinicopathologic effects of BRAF in this cancer type remain to be fully elucidated.

ERBB2 encodes HER2, a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. ERBB2 mutations have been reported in ~12% of urothelial carcinomas. This particular variant I767M lies within the protein kinase domain of HER2 and has been shown to result in increased kinase activity in vitro. This variant has been identified in rare cases of urothelial carcinomas, among other cancer types, and is considered to be oncogenic. Further studies are needed to elucidate the role of anti-HER2 therapy in this setting. Clinical correlation is recommended.

FBXW7 is a tumor suppressor gene responsible for the degradation of several proto-oncogenes, and is mutated in several tumors including colorectal, liver, bladders and ovarian cancers. mTOR is one of the substrates of FBXW7-mediated protein degradation, and loss of function of FBXW7 increases the levels of total and activated mTOR. FBXW7 loss of function mutations have been described in many types of adenocarcinoma including colon, bladder and lung. FBXW7 mutations are present in approximately 7% of urothelial carcinomas. Preclinical data have suggested that inactivating mutations of FBXW7 could predict sensitivity to the mTOR inhibitor rapamycin. However, this particular variant (H500Y) has not been characterized in the scientific literature. Therefore, the effect on protein function is unknown. Clinical correlation is recommended.

ATM alterations have been reported as germline variants which predispose to inherited cancer syndromes and as somatic (acquired) variants in tumors. ATM is part of many signalling networks, including cell metabolism and growth, oxidative stress, and chromatin remodelling, all of which can affect cancer progression. Although ATM is considered to be a tumour suppressor, ATM signaling may be advantageous to cancer cells in some settings, particularly in resistance to radio- and chemotherapeutic treatment. For this reason, the use of ATM inhibitors in cancer therapy is under exploration. Genetic alterations in ATM have been reported in ~15% of urothelial carcinomas. However, the significance of this particular variant (R2443Q) is unknown, according to ClinVar (https://preview.ncbi.nlm.nih.gov/clinvar/variation/142211/)

PIK3CA mutations activate the PI3K-PTEN-AKT pathway which is downstream from both the EGFR and the RAS-RAF-MAPK pathways. PIK3CA mutations are present in ~5% of cutaneous melanomas. PIK3CA M1043I is a known oncogenic hotspot mutation. M1043I confers a gain of function on the protein as indicated by in increased activation of downstream signaling and and transformation in cell culture. Clinical trials targeting PI3K/Akt/mTor pathway inhibitors are available for patients with PIK3CA mutated tumors.

PTEN is an obligate haplo-insufficient tumor suppressor gene and is mutated in a large number of cancers. It encodes a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/mTOR signaling pathway. PTEN mutations are loss-of-function mutations and occur in 3% of urothelial carcinomas. The above variants (R130Q and G165R) are predicted to result in loss of function based on preclinical in vitro studies. Whether PTEN alterations predict for responsiveness to mTORC1 inhibitors is less certain at this time.

JAK3 is a non-receptor protein tyrosine kinase involved in the interferon-alpha/beta/gamma pathway and is a member of the JAK/STAT signaling pathway. The JAK3 V722I variant has been reported as a likely benign germline polymorphism (ClinVar, https://preview.ncbi.nlm.nih.gov/clinvar/variation/134573/) and also as an acquired somatic variant in some tumors. It has been reported to be an activating variant of JAK3 and initial in vitro studies suggest that this variant may play a role in the regulation of PD-L1 expression. Also, V722I resulted in constitutive phosphorylation of Jak3 and was transforming in cell culture. Clinical correlation is recommended.