Immunotherapy is revolutionizing bladder cancer treatment, particularly in platinum-unfit patients.4 Promising trials such as PURE-01, ABACUS, and NABUCCO showed substantial pathological complete response rates with neoadjuvant immunotherapy.4 However, predicting treatment response remains an unmet clinical need, prompting ongoing investigations for novel biomarkers.
Molecular and histological classifications have been explored, recognizing UC's heterogeneity with high tumor mutational burden (TMB) and genomic instability. Genomic sequencing, using techniques such as next-generation sequencing (NGS), may potentially play a key role in the diagnostic, staging, and therapeutic processes, enhancing clinical outcomes.5 The evolving genomic characterization of bladder tumors reveals distinct biological subtypes with varied mutational landscapes and related prognoses, opening avenues for targeted treatments. However, implementing complete gene sequencing for all patients in everyday practice can be challenging due to time, costs, and equipment constraints.5 Several papers explored alternative, cost-effective techniques like immunohistochemical analysis (IHC), in situ hybridization (ISH), and real-time PCR (qPCR).6,7
Our review focused on the evolution of molecular classifications in bladder cancer, evaluating their potential therapeutic implications.
In 2012, Sjodahl et al. examined transcriptome data from 308 UC samples, identifying five distinct subtypes according to the different expression of cell-cycle and cell adhesion genes and various cytokeratins.8 “Urobasal A” exhibited elevated expression of FGFR3, CCND1, and TP63, along with genes related to the early cell cycle, showing a favourable prognosis. “Genomically unstable” displayed strong immune features, late cell-cycle genes, TP53 mutations, and ERBB2 expression. “Urobasal B”, “squamous cell carcinoma-like” and “infiltrated” subtypes expressed immunological signature genes, with the latter two showing the worst prognosis, according to the authors.8
In 2014, Choi and colleagues, following the line of previous studies on breast cancers, conducted a whole-genome mRNA expression profiling on 73 BC patients from MD Anderson Cancer Center (MDA) proposing a three-tiered classification.9 The “luminal” subtype exhibited enrichment in CDH1, epithelial biomarkers, peroxisome proliferator activated receptor gamma (PPAR-γ), estrogenic receptor, and FGFR3 activating mutations, demonstrating a promising 54% response to NAC. “P53-like” tumors, with a comparable transcriptomic profile to luminal subtype but with activated wild-type p53 and CDKN2A, showed complete NAC resistance (10% of responses, in their series). Lastly, the “basal” subtype, marked by squamous differentiation and p63 expression, displayed aggressive behaviour and poor prognosis, when compared with the others.9
During the same year, Damrauer et al. developed BASE47, a 47-gene predictor, differentiating high-grade UCs into luminal and basal-like tumors.10 “Luminal” subtypes exhibited urothelial differentiation markers, such as UPK1B, UPK2, and UPK3A, while “basal-like” tumors expressed stemness markers, RB1, and TP53 mutations, which reflected in a worse prognosis. Furthermore, Damrauer's model suggests different tumorigenesis patterns for low-grade and high-grade tumors, emphasizing specific biomolecular alterations in each subtype. For example, basal-like tumors, more common in females, may have a distinct pathogenesis, potentially linked to chronic inflammation.10
Subsequently, the TCGA project analyzed 131 high-grade MIUC, incorporating comprehensive assessments of DNA, RNA, microRNA, protein expression, DNA methylation, and more. The RNA-based analysis identified four categories (called “clusters” I-IV).11 The tumors were classified by two frequently altered pathways: PI3K-AKT-mTOR signaling and tyrosine kinase receptors/RAS. Alterations in these pathways were found in 42% and 44% of tumors, respectively, suggesting therapeutic implications. The study reported a high somatic mutation rate, analogous to non-small cell lung cancer and melanoma, hinting at potential efficacy with immune checkpoint inhibitors.11
In 2017, Seiler and colleagues assessed the impact of molecular subtypes in 343 MIBC patients on NAC response and survival.12 “Basal-like” tumors derived significant benefits from NAC due to their highly proliferative nature. “Luminal-infiltrated” subtype, associated with advanced stages, had poorer outcomes, while “luminal non-immune-infiltrated” tumors showed the best prognosis, seemingly unresponsive to chemotherapy. Finally, “claudin-low” subtype exhibited a mesenchymal signature, high immune infiltration, and poor prognosis, with disappointing responses to immune checkpoint inhibitors. Molecular subtypes' impact on immunotherapy response remains uncertain, according to this classification.12
In the same years, TCGA project proposed a stratification of BCs into five subtypes.13 “Luminal-papillary” tumors, characterized by FGFR3 mutations and papillary histology, suggest potential efficacy with FGFR3-targeted inhibitors. “Luminal-infiltrated” subtype, with high heterogeneity and immune marker expression, responds to immune checkpoint inhibitors, although conflicting results on predictors like PD-L1 exist. The “luminal subtype” lacks a defined therapeutic approach, while “basal-squamous” responds well to both cisplatin-based NAC and immune checkpoint inhibitors, and the “neuronal subtype” suggests etoposide-cisplatin therapy.13
Kamoun et al.'s 2020 classification of 1750 MIBC samples identified six molecular subtypes, each one with distinct genetic mutations and transcriptomic profiles.14 For instance, “luminal papillary” tumors showed FGFR3 mutations, while “luminal non-specified” had PPAR-γ amplifications. The “luminal unstable” subgroup was enriched in TP53 and excision repair cross-complementation group 2 (ERCC2) mutations while the “stroma-rich” subtype exhibited overexpression of various gene signatures. The “basal/squamous-like” tumors were characterized by TP53 mutations. The “neuroendocrine-like” subtype had the worst prognosis.14
In 2020, Taber et al. conducted a comprehensive multi-omics analysis on 300 bladder cancer patients, aiming to identify molecular markers predicting responses to cisplatin-based chemotherapy.15 High genomic instability, characterized by chromosomal alterations, single-base substitution 5 (SBS5), indels, and BRCA2 mutations, correlated with a better response rate (71% vs. 49%, high vs. low genomic instability). Transcriptomic analysis revealed that the “Basal/Squamous” (Ba/Sq) subtype had a lower response rate. Combining Ba/Sq subtype with genomic instability identified distinct response patterns, with 90% response in high genomic instability non-Ba/Sq subtype, and 20% response in low genomic instability Ba/Sq subtype. Proteomics analysis linked immune cell infiltration and high PD-1 expression to treatment response.15
In 2022, Sjodahl and colleagues applied molecular subtype classification to 149 BC patients undergoing neoadjuvant or induction chemotherapy [16]. Subtypes included “urothelial” (UroA, UroB, UroC), “genomically unstable” (GU), “basal/squamous” (Bs/Sq), “mesenchymal-like” (Mes-like), and “small cell/neuroendocrine-like” (Sc/NEU). Neoadjuvant-treated patients with GU and Uro subtypes exhibited higher complete response rates (52% GU, 31% Uro) than Bs/Sq subtype (21%). Radical surgery alone did not show significant subtype associations with pathological response or prognosis. Comparison with prior studies indicated similarities in subtype distribution and identified genes like SPP1, HOXD10, and PARP6 with lower expression in complete responders, emphasizing potential response-associated molecular markers.16
In conclusion, various immunohistochemistry and molecular biomarkers have been explored to predict neoadjuvant treatment response in bladder cancer, but no validated markers exist for routine clinical use. Recent efforts have focused on identifying biomarkers for MIBC to guide therapies and improve prognosis. Furthermore, molecular sequencing can aid in identifying non-responders to NAC, preventing ineffective treatments and unnecessary side effects.
Among the others, FGFR3 mutations, prominent in luminal papillary tumors, suggest the potential efficacy of FGFR inhibitors, benefiting a subset of advanced and metastatic cases.9,13,14
Immune checkpoint inhibitors (ICIs) like anti-PD1/PD-L1 are standard therapies for advanced cancers, but their role in early-stage tumors remains unclear. PD-L1 expression and TMB have been inconsistent predictors of ICI response; potential other biomarkers for predicting ICI response may include CD8+ lymphocytes and proinflammatory cytokines. As previously analyzed, subtypes with "hot" and immunogenic microenvironments, like basal/squamous and luminal-infiltrated, may benefit from ICI therapy alone or in combination with chemotherapy.14,15
DNA damage response and repair (DDR) gene alterations, observed in 23-54% of bladder cancer patients, suggest sensitivity to cisplatin-based therapies and immunotherapy. ERCC2 mutations, a subtype of DDR gene alteration, correlated with favourable responses to cisplatin-based NAC.14
Despite progress, UC remains challenging, necessitating further efforts to validate predictive biomarkers and apply molecular classifications in clinical practice, despite potential complexities and costs associated with whole transcriptome profiling for all patients.
Written by: Nicole Conci,1,2 Elisa Tassinari,1,2
- Medical Oncology, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy.
- Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy.
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- Massari F, Santoni M, Di Nunno V, Cheng L, Lopez-Beltran A, Cimadamore A, et al. Adjuvant and neoadjuvant approaches for urothelial cancer: Updated indications and controversies. Cancer Treat Rev. 2018 Jul;68:80-85.
- Pfister C, Gravis G, Fléchon A, et al. Dose-Dense Methotrexate, Vinblastine, Doxorubicin, and Cisplatin or Gemcitabine and Cisplatin as Perioperative Chemotherapy for Patients With Nonmetastatic Muscle-Invasive Bladder Cancer: Results of the GETUG-AFU V05 VESPER Trial. J Clin Oncol. 2022;40(18):2013-2022.
- Chen H, Yang W, Xue X, Li Y, Jin Z, Ji Z. Neoadjuvant immunotherapy and chemoimmunotherapy for stage II-III muscle invasive bladder cancer. Front Immunol. 2022; 13:986359.
- Fantini D, Meeks JJ. Genomic classification and risk stratification of bladder cancer. World J Urol. 2019;37(9):1751-1757.
- Font A, Domènech M, Benítez R, Rava M, Marqués M, Ramírez JL, et al. Immunohistochemistry-Based Taxonomical Classification of Bladder Cancer Predicts Response to Neoadjuvant Chemotherapy. Cancers (Basel). 2020;12(7):1784.
- Helal DS, Darwish SA, Awad RA, Ali DA, El-Guindy DM. Immunohistochemical based molecular subtypes of muscle-invasive bladder cancer: association with HER2 and EGFR alterations, neoadjuvant chemotherapy response and survival. Diagn Pathol. 2023;18(1):11.
- Sjodahl G, Lauss M, Lovgren K, Chebil G, Gudjonsson S, Veerla S, et al. A molecular taxonomy for urothelial carcinoma. Clin Cancer Res. 2012;18: 3377–86.
- Choi W, Porten S, Kim S, Willis D, Plimack ER, Hoffman-Censits J, et al. Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy. Cancer Cell. 2014;25(2):152-165.
- Damrauer JS, Hoadley KA, Chism DD, Fan C, Tiganelli CJ, Wobker SE, et al. Intrinsic subtypes of high-grade bladder cancer reflect the hallmarks of breast cancer biology. Proc Natl Acad Sci U S A. 2014;111(8):3110-5.
- Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014;507(7492):315-22.
- Seiler R, Ashab HAD, Erho N, van Rhijn BWG, Winters B, Douglas J, et al. Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy. Eur Urol.2017; 72(4):544-554.
- Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell. 2017; 171(3):540-556.e25.
- Kamoun A, de Reyniès A, Allory Y, Sjödahl G, Robertson AG, Seiler R, et al. A Consensus Molecular Classification of Muscle-invasive Bladder Cancer. Eur Urol. 2022;77(4):420-433.
- Taber A, Christensen E, Lamy P, Nordentoft I, Prip F, Lindskrog SV, et al. Molecular correlates of cisplatin-based chemotherapy response in muscle invasive bladder cancer by integrated multi-omics analysis [published correction appears in Nat Commun. 2022 Apr 4;13(1):1916]. Nat Commun. 2020;11(1):4858. doi:10.1038/s41467-020-18640-0
- Sjödahl G, Abrahamsson J, Holmsten K, Bernardo C, Chebil G, Eriksson P, et al. Different Responses to Neoadjuvant Chemotherapy in Urothelial Carcinoma Molecular Subtypes. Eur Urol. 2022;81(5):523-532.