Non–muscle-invasive bladder cancer (NMIBC) is heterogeneous, but current diagnostic and treatment strategies rely primarily on clinical parameters, lacking individualization to tumor and host genetics and biology. The heterogeneity of NMIBCs is derived from mutations, mutation signatures, chromosomal loss, and disruption of molecular pathways, which ultimately affects tumor progression, recurrence, and responsiveness to intravesical and systemic chemotherapy. Although research is still underway, advances in sequencing technology, insight into differential bacillus Calmette-Guérin responses, and new investigational treatment targets will soon offer clinicians new, precision-based tools to risk stratify and determine treatment regimens for future patients with bladder cancer.
Key points
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Non–muscle-invasive bladder cancers (NMIBCs) are heterogeneous across stage and grades. The current treatment relies on clinical and pathologic parameters.
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Much less is known about the genetic features of NMIBC compared with muscle-invasive bladder cancer, and these discoveries may help identify targeted therapies.
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Ongoing investigations into immunotherapies for NMIBC are critical given shortages and differential responsiveness to bacillus Calmette-Guérin.
Introduction
Urothelial carcinoma (UC) is the fifth most common cancer in the United States. Of diagnosed patients, nearly 80% present with non–muscle-invasive bladder cancer (NMIBC), comprising tumor stages Ta, T1, and carcinoma in situ (CIS). Each stage of NMIBC has recently been found to have distinct biological and molecular features, which may translate into clinical heterogeneity affecting recurrence, progression, and response to therapy. Stage Ta tumors include papillary urothelial neoplasms of low malignant potential and low-grade and high-grade noninvasive papillary UC, which are predominated by luminal differentiation. Few Ta tumors progress to muscle-invasive disease, but recurrence rates are high, requiring frequent intravesical therapy. Alternatively, invasive cancers (stage T1) and preinvasive CIS are more likely to progress to muscle invasion and metastasis. Although most T1 tumors are high grade, all CIS is considered high grade and usually responsive or partially responsive to bacillus Calmette-Guérin (BCG) immunotherapy.
The current treatment of NMIBC relies on clinical and pathologic parameters largely based on stage and grade, lacking individualization to tumor and host biology. However, recent advances in genomics technology pioneered by The Cancer Genome Atlas (TCGA) have facilitated the comprehensive analysis of genomic, epigenetic, and transcriptomic alterations across tumor types. , These advances, coupled with ongoing research into new chemotherapeutic strategies for NMIBC, are intended to individualize future cancer treatment. This article highlights key features of tumor and host biology that may be applied for the risk stratification and treatment of NMIBC in this new era of precision medicine.
Genomic landscape of non–muscle-invasive bladder cancer
The heterogeneity of NMIBCs is derived from mutations, mutation signatures, chromosomal loss, and disruption of molecular pathways. Fig. 1 A and B compare the heterogeneity of stage Ta and T1, as well as differential gene expression profiles of tumors with recurrence compared with nonrecurrence. ,
Apolipoprotein B Messenger RNA Editing Enzyme Catalytic Polypeptidelike–Mediated Mutagenesis and Intratumor Heterogeneity
DNA is constantly under the stress of mutation and repair. Within a tumor, patterns of mutations may develop, termed a signature of this mutagenesis. These mutation signatures vary by age, tumor type, and origin of cancer. In bladder cancer, the predominant mutation signature is apolipoprotein B messenger RNA editing enzyme catalytic polypeptidelike (APOBEC) mediated. APOBEC are a family of cytosine deaminases, which under physiologic conditions perform C>U editing of single-stranded DNA. During cytosine deamination, APOBEC catalyzes a 5′-T-phosphate-C-3’ (TpC) to T, G, or A, which, if unregulated, can lead to hypermutation. , , Therefore, in bladder cancer APOBEC activity has been associated with a high tumor mutation burden (TMB). TCGA 2014 and 2017 analyses of muscle-invasive bladder cancer (MIBC) specimens showed APOBEC mutation patterns in 93% and 67%, respectively. , An increase in TMB reflects genomic stress but may be a good prognostic biomarker because MIBCs with higher TMB were associated with improved survival. When low APOBEC MIBCs were evaluated, our group identified an enrichment of oncogenes RAS and FGFR3 , suggesting that processes driving APOBEC-mediated mutagenesis were complimentary to oncogene-induced tumor growth. However, in NMIBC the TMB is at least 3-fold lower compared with MIBC and enrichment of APOBEC signatures increases with stage and grade. , In an analysis of 140 Ta tumors, high-grade (GS2) tumors were more likely to be APOBEC enriched with high rates of recurrence. Similarly, a prospective European multi-center collaboration (FP7:UROMOL), showed an association between high-risk NMIBC and APOBEC mutagenesis. Therein, class 2 tumors, which showed worse progression and higher rates of concomitant CIS, had increased APOBEC mutation signatures ( P = 3.4 × 10 −8 ) and expression of APOBEC3A and 3B . In addition, APOBEC expression may vary over the lifetime of a tumor, especially in tumors with progression. , In a small cohort of 29 patients with serial biopsies, Lamy and colleagues found higher variance in TMB and increased APOBEC enrichment in patients who progressed (53%, 8 out of 15) compared with nonprogressors (7%, 1 out of 14) ( P <.009). Whether tumors that progress have higher genomic instability with or from APOBEC-mediated repair is unknown.
Chromosomal Aberrations
Increase in copy number alterations and genomic instability is linked to higher stage and worse prognosis in bladder cancer. Furthermore, systematic loss of chromosome regions has been identified in patients with NMIBC, which have direct implications on prognosis. , , Although most NMIBCs are diploid or near diploid, loss of specific regions is associated with higher recurrence. , , A comparison of genetic aberrations in 28 Ta versus 28 T1 tumors found loss of 9q (54%), 9p (39%), Y (28%), and gain of 1q (14%) were more prevalent in Ta tumors, whereas loss of 2q (36%), 8p (32%), and 11p (21%) was more common in T1 tumors. Hurst and colleagues similarly reported loss of chromosome 9q in 41% of Ta tumors resulting in the supervised clustering of Ta tumors into GS1 (normal for 9q) or GS2 subtypes (loss of 9q). GS2 tumors were predominantly high grade ( P = .0053), with 9q deletion present in 3.4% GS1 versus 87.3% GS2. Interestingly, loss of 9q has also been shown in normal urothelium adjacent to areas of tumor. This finding suggests loss of 9q may be an early marker of local genomic instability and may play a role early in the initiation of bladder cancer development. ,
Candidate tumor suppressor genes on chromosome 9q that may play a role in bladder cancer pathogenesis are NOTCH1 and TSC1 . NOTCH1 , which acts predominantly as a tumor suppressor compared with family members NOTCH2 and 3 , has been shown to be downregulated in bladder cancer, with inactivation of NOTCH1 occurring more frequently in high-grade tumors. Rampias and colleagues found Notch pathway or NOTCH1 mutation in 61% (44 out of 72) and 51% (37 out of 72) of bladder cancer specimens, respectively, with presence of mutation significantly correlating with risk of death ( P = .059) and muscle-invasive disease ( P = .037). Loss of TSC1 , the negative regulator of mammalian target of rapamycin (mTOR), may be associated with increased cell growth and survival in NMIBC, and gene expression studies of GS2 tumors suggest increased activation of mTOR pathways in high-risk disease. TSC1 would be an exciting target for NMIBC because everolimus may be a cost-effective therapy to prevent recurrence of GS2 tumors. Although everolimus has not been universally effective in metastatic urothelial cancer, unique responders with alterations in TSC1 and NF1 have been identified to have durable response. Furthermore, a recent pilot study of rapamycin after cystectomy showed enhanced immune activity that may decrease tumor recurrence.
RTK-RAS–Phosphatidylinositol 3-kinase Pathway (Fibroblast Growth Factor Receptor 3, ERBB2 (Erb-b2 Receptor Tyrosine Kinase 2), Cancer-Associated Phosphatidylinositol 3-kinase)
Fibroblast growth factor receptor 3
NMIBCs have features of differentiated urothelium with consistent activation of the fibroblast growth factor receptor 3 (FGFR3) and Ras pathways. FGFR3 signals through Ras (RAS-MAPK-ERK pathway) and regulates cell cycle entry and proliferation. Mutation in FGFR3 is most common in Ta tumors (∼80%), with decreased frequency noted in high-grade Ta (59%), T1 (10%–34%), and MIBC (10%–20%) ( Fig. 1 C). A pooled meta-analysis by Liu and colleagues reiterated these findings, showing increased frequency of FGFR3 mutation in low-grade (Relative Risk (RR), 2.948; 95% confidence interval [CI], 2.357–3.688) and early-stage (RR = 2.845; 95% CI = 2.145–3.773) bladder cancer as well as an association with improved disease-free, progression-free, and recurrence-free survival. Of those NMIBCs that progress to MIBC, mutation of FGFR3 was found in 25% of tumors compared with only 7% of tumors that presented as MIBC.
FGFR3 fusion proteins are also implicated in bladder cancer pathogenesis, with FGFR3-TACC3 fusions being the most common. TACC3 is upstream of FGFR3 signaling and this fusion protein causes constitutive activation of the MAPK-ERK pathway, unregulated cell proliferation, and promotion of aneuploidy. Although more commonly associated with MIBC, FGFR3-TACC3 fusions have been identified in NMIBC cell lines and reported in 1 Ta tumor (1 out of 23, low-grade Ta) by Pietzak and colleagues. , , Small molecule inhibitors targeting FGFR3 have been used in metastatic bladder cancer. Although dovitinib was considered to be unsuccessful for BCG-refractory bladder cancer, with a complete response rate of only 8%, targeting tumors with FGFR3 alterations has been associated with improved response. In a phase II trial of erdafitinib for metastatic UC with FGFR3 alterations, the overall response rate was 40% and, of those treated with 8 mg, 76% had a reduction in size of their largest tumor. However, the median duration of response was only 5.6 months (4.2–7.2 months). In a similar study of patients with metastatic UC with FGFR3 alterations, a pan-FGFR–blocking antagonist, BGJ398, was administered in a 3-weeks-on, 1-week-off cycle. The overall response rate was 25.4%, with disease stabilization in 38.8% and response duration of 5.06 months (95% CI, 3.91–7.36 months). Interestingly, FGFR3 mutations were identified by cell-free DNA in 68% of patients, which could translate to methods of sampling tumor or urine without the need for recurrent biopsy. In addition, there are 2 additional clinical trials assessing FGFR3 blockade in patients with metastatic UC harboring FGFR3 mutations ( NCT02365597 , NCT02038673 ). Although most published results on FGFR3 alterations are in metastatic disease, the high frequency of FGFR3 mutation in NMIBC suggests that FGFR3 may be a rational target in NMIBC as well depending on the tolerability of each therapy.
ERBB2
ERBB2 encodes human epidermal growth factor receptor 2 (Her2), which is mitogenic for cell growth and survival. Of all cancer types, ERBB2 amplification occurs third most commonly in bladder cancer and is associated with high-risk features including risk of progression and concomitant CIS in NMIBC specimens. Pietzak and colleagues characterized genomic alterations in 105 NMIBCs (12 high-grade Tis, 23 low-grade Ta, 12 high-grade Ta, and 28 high-grade T1) and found ERBB2 mutations were only present in high-grade NMIBC tumors apart from a single low-grade Ta tumor. ERBB2 mutations were associated with higher-stage (HGT1, ∼25%; HGTa, ∼20%; LGTa, ∼5%; P = .05) and high-grade (HGTa, ∼20% vs LGTa, ∼5%%; P = .01) tumors compared with FGFR3 mutations, which were present in only 39% of high-grade tumors and more characteristic of low-grade, low-stage NMIBC (LGTa, ∼80%; HGTa, ∼60%; HGT1 ∼35%). In UROMOL, ERBB2 mutations were more common in class 2 tumors and were altered in 20% of tumors. These findings suggest FGFR3 alterations may be more common in low-grade, noninvasive tumors, whereas ERBB2 alteration is a driver of higher-stage and higher-grade tumors. The authors examined the expression across Ta and T1 (see Fig. 1 C) and found little association with stage.
Cancer-associated phosphatidylinositol 3-kinase
The phosphatidylinositol 3-kinase (PI3K)/mTOR/protein kinase B (AKT) pathway may provide growth advantage during the initiation of cancer. PIK3CA (cancer-associated phosphatidylinositol 3-kinase) encodes the catalytic subunit of PI3K and is more frequently mutated in NMIBC compared with MIBC (Ta, 40%–50% ; T1, 6%–20% ; MIBC, 22% ). , , Hurst and colleagues , found that 40% to 50% of Ta tumors and 6% to 20% of T1 tumors harbored a PIK3CA mutation, which typically occurred as a point mutation in a codon critical for catalytic activity. The UROMOL series identified mTOR/PI3K alterations in 59% of tumors with NF1 mutations in 24%, PIK3CA in 23%, and PIKR in 9% of patients with mutations in PIK3CA commonly coexisting with FGFR3 mutations. , , Our evaluation of 25 T1 tumors did not find a significant difference in frequency of PIK3CA mutations in high-risk NMIBC specimens (T1 high grade or Ta high grade with CIS) from patients who progressed to muscle-invasive or metastatic stages compared with nonprogressors. Collectively, this finding suggests this mutation may play a role early in bladder cancer initiation or recurrence rather than disease progression.
Chromatin-Modifying Genes (ARID1A, KDM6A) and Epigenetic Dysregulation
Chromatin-modifying genes (CMGs) are commonly mutated across cancer types and serve as regulators of gene expression. , Pietzak and colleagues found that the 2 most commonly mutated CMGs in NMIBC were KDM6A (38%) and ARID1A (28%). Although neither mutation correlated statistically with tumor stage or grade, KDM6A mutation frequency decreased with increasing grade/stage (52% LGTa, 38% HGTa, 32% HGT1, 25% Memorial Sloan Kettering (MSK)-MIBC, 24% TCGA-MIBC), whereas ARID1A mutation frequency tended to increase with increasing grade/stage (9% LGTa, 28% HGTa, 18% HGT1, 30% MSK-MIBC, 24% TCGA-MIBC). An increased frequency of KDM6A mutations was found in female patients with Ta tumors (20 out of 27, 72%) compared with men (23 out of 55, 42%; P = .0092). KDM6A is not X inactivated in women and the increased frequency of mutations may suggest increased pressure on KDM6A in women with 2 expressed copies. Tumors with mutations of KDM6A may have increased H3K27me3 from unbalanced epigenetic regulation at H3K27. To counter increased H3K27me3, treatment with an EZH2-inhibitor may normalize enhancer methylation. Our research group has also found increased sensitivity to EZH2 inhibitors in tumors with KMT2C alteration that disrupts recruitment of KDM6A (under investigation).
ARID1A mutation has been associated with increasing stage (24% MIBC vs 20% NMIBC) and aggressiveness. , Pietzak and colleagues reported that alterations in ARID1A were significantly associated with increased risk of recurrence, which may correspond with increased aggressiveness or BCG-resistance (hazard ratio [HR], 3.14; 95% CI, 1.51–6.51; P = .002). With further investigation, ARID1A may serve as a predictive biomarker in patients undergoing BCG therapy or a potential therapeutic target to enhance BCG response (discussed later).
Telomerase Reverse Transcriptase Promoter
Enzymatic activation of telomerase maintains the 3′ telomere length at the ends of chromosomes to avoid senescence and apoptosis and promote cellular lifespan. Promoter mutations in the telomerase reverse transcriptase gene ( TERT ) are found in many human cancers (eg, melanoma, glioblastoma, hepatocellular carcinoma), including both NMIBC and MIBC. , , , Pietzak and colleagues identified TERT promoter mutations in 61% of LGTa, 88% of HGTa, 79% of HGT1, and 85% of MSK-MIBC tumors without a significant difference across stage ( P = .2) or grade ( P = .15), with similar frequencies noted in other studies. Given its high frequency of occurrence and persistence across various bladder cancer grades/stages, TERT may play a functional role in early bladder tumorigenesis. , A common polymorphism within the TERT promoter binding site acts as a modifier of the TERT mutation and affects survival and tumor recurrence. In the absence of this variant allele polymorphism, patients with Tis, Ta, and T1 tumors bearing TERT mutations trended toward worse survival (HR, 2.19; 95% CI, 1.02–4.70) and higher risk of tumor recurrence (HR, 1.85; 95% CI, 1.11–3.08) compared with patients with the allele polymorphism present. From a diagnostic standpoint, TERT promoter mutations are easily detected in voided urine and could aid in risk stratification of patients early in disease. ,
Genomics of Tumor Progression
Although most patients diagnosed with bladder cancer are non–muscle invasive at presentation, ∼15% to 20% of patients with NMIBC progress to muscle invasive (MIBC). Studies have referred to these NMIBCs as progressors or secondary MIBC. Two of the candidate genes proposed in tumor progression are E2F1 and CDKN2A . E2F1 is a regulator of cell apoptosis and has been linked with tumor invasion and metastasis in multiple cancer types. Microarray gene expression profiling of 102 NMIBCs showed upregulation of E2F1 and its downstream targets, EZH2 and SUZ12 , in patients with progression to muscle-invasive disease ( P <.001). Transfection of urothelial cells with a plasmid vector coding E2F1 , EZH2 , and SUZ12 found that their expression correlated with increased proliferation, migration, invasiveness, and chemoresistance to mitomycin C and BCG. CDK2NA is a cell cycle regulator involved in G1-S cell cycle arrest. CDKN2A was found to be lost in the invasive portion of NMIBCs and our group identified loss of CDKN2A in 37% of T1s at progression compared with only 6% in nonprogressors ( P = .10). , Only tumors with progression had both TP53 and CDK2NA loss, suggesting that loss of both checkpoints in cell cycle progression may be necessary for progression.
In a comparison of primary with secondary MIBC, the frequency of ERCC2 mutations was significantly greater in primary MIBCs (11% vs 1.8%; P = .044), potentially resulting in decreased response to cisplatin chemotherapy (26% vs 45%; P = .02) and significantly worse recurrence-free, cancer-specific, and overall survival. Whether ERCC2 mutations are more common in primary MIBC, or loss of ERCC2 mutations occurs during clonal selection and progression to MIBC is unknown. One hypothesis to explain the aggressive nature of secondary MIBC is that during progression TMB decreases to reduce the number of neoantigens, resulting in immune escape. Although more research is needed, insight into progression genomics is critical because patients with NMIBC with risk factors for progression may benefit from upfront cystectomy or enrollment in clinical trials rather than standard immunotherapies (eg, BCG) at the time of initial diagnosis.
Predicting response to bacillus Calmette-Guérin
BCG is the most effective intravesical immunotherapy available for NMIBC to decrease recurrence (32.6%–42.1%), progression (9.5%–13.4%), and death from bladder cancer, but at least 40% of patients do not respond to BCG, and response decreases with age. Tumor, host, and BCG factors are identified here that may aid in predicting BCG responsiveness and risk stratification before and while on BCG therapy. Known clinicopathologic factors that predict recurrence after BCG therapy include female sex, tumor multiplicity, and presence of CIS, whereas high-grade disorder, T1 tumors, and early recurrence on 3-month endoscopic evaluation predict progression to MIBC. Upfront limitations in overall BCG data interpretation include differences in BCG strains; maintenance BCG protocol; and definitions of clinical response, recurrence, progression, and failure.
Tumor Factors
Neoantigens
Self-antigens are expressed by both tumor and normal cells, which leads to tolerance by the immune system to these antigens. Neoantigens derived from mutated proteins are specific to cancer cells only, which limits tolerance, promotes immune cell infiltration, and makes these proteins attractive candidates for immunotherapy targets. Neoantigens are prognostic and are associated with overall survival in MIBC. In lung cancer and melanoma, tumor responsiveness to immune checkpoint inhibitors is directly correlated with increased TMB and neoantigen load, with the top 20% of TMB being more likely to respond. Thus, tumors with a low TMB may have a decreased response to BCG. Our group showed a significant decrease in TMB and thus neoantigen load in patients who progressed on BCG or had metastatic disease compared with nonprogressors ( P = .02). In a comparison of 35 NMIBCs treated with BCG (17 responsive and 18 unresponsive), the median TMB was 3 mutations per mutation burden (MB), with a significant difference noted in responsiveness (4.9 mutations per MB vs 2.8 mutations per MB; P = .017). Tumors with a high TMB (>3 mutations per MB) were associated with a greater response to BCG (71% vs 28%; P = .01). Higher TMB was associated with longer recurrence-free survival (38 vs 15 months; P = .0092). Furthermore, loss of TMB was most recently found in the PURE-01 cohort in tumors that progressed. These findings suggest that increased TMB may be predictive of responsiveness to BCG, and this possibility continues to be investigated.
Genomics of bacillus Calmette-Guérin responsiveness
Genetic features associated with BCG response is an active area of research. Table 1 summarizes current knowledge regarding specific genes associated with BCG responsiveness in NMIBC. Ke and colleagues found that certain glutathione (GSH) pathway genomic variations could predict recurrence after BCG. GSH is involved in cellular antioxidation and detoxification as well as T-cell and neutrophil function and survival. In 191 patients with NMIBC who underwent BCG therapy (induction ± maintenance), recurrence after BCG was significantly associated with polymorphism rs7265992 in GSH synthetase ( GSS ). As discussed earlier, our group found that in 10 patients with high-risk NMIBC treated with BCG, predictors of progression to MIBC included high TMB and loss of CDK2NA . Pietzak and colleagues found ARID1A mutation (a chromatin-modifying gene) was significantly associated with increased risk of recurrence (HR, 3.14; 95% CI, 1.51–6.51; P = .002) after BCG induction in 62 patients with high-grade NMIBC. Lima and colleagues developed a BCG responsiveness predictive score using clinicopathologic factors as well as immune system gene polymorphisms from genotyping serum of 204 patients with NMIBC treated with BCG (single nucleotide polymorphisms in tumor necrosis factor α [TNFA]-1031T/C [rs1799964], interleukin [IL] 2 receptor α [IL2RA] rs2104286 T/C, IL17A-197G/A [rs2275913], IL17RA-809A/G [rs4819554], IL18R1 rs3771171 T/C, intercellular adhesion molecule 1 [ICAM-1] K469E [rs5498], Fas ligand [FASL]-844T/C [rs763110], and tumor necrosis factor [TNF]-related apoptosis-inducing ligand receptor 1 [TRAILR1]-397T/G [rs79037040]). In addition, Kim and colleagues identified 424 and 287 genes that were significantly associated with recurrence and progression-free survival, respectively, in 80 T1 patients after BCG therapy. From these, they identified gene signatures predictive of recurrence (12 total; HR, 3.38; P = .048) and progression (12 total; HR, 10.49; P = .048) (see Table 1 ).
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