Advances in molecular classification of bladder cancer

LIU Zhi-tao, LIU Fu-jin, WANG Fei?

1. Department of Urology, Affiliated Hainan Hospital of Hainan Medical University, Hainan General Hospital, Haikou 570311, China

2. Department of Pathology, Affiliated Hainan Hospital of Hainan Medical University, Hainan General Hospital, Haikou 570311, China

Keywords:

ABSTRACT Bladder cancer is the second most common tumor in the urinary system after prostate cancer.It is highly heterogeneous and its developmental mechanism involves abnormal alterations in the structure and function of multiple genomes.Researching the molecular classification of bladder cancer by using molecular biology techniques is important for defining the pathogenesis of the disease and selecting therapeutic schedule.This paper will review the progress of molecular classification studies of bladder cancer.

1.Molecular classification of NMIBC

At present, there are not many researches and reports on molecular classification related to NMIBC.With the further research of MIBC,the industry has gradually strengthened its attention to NMBIC,in order to bring more efficient diagnosis and treatment for related patients.At present, the mainstream molecular classification of NMIBC is the UROMOL classification proposed by Hedegaard et al.[5] in 2016.Researchers conducted transcriptome sequencing analysis on 476 samples (460 NMIBC, 16 MIBC) from multiple centers, and divided NMIBC into three categories: Class 1, Class 2 and Class 3.They also found that Class 1 was similar to Urobasal A (Uro A) proposed by Sjodahl and colleagues.By analyzing the mixed cohort of NMIBC and MIBC, Uro A was found in most NMIBC[6].Class 2 highly expressed late cell cycle regulators and ERBB2-related transcription factors, and Class 2 was similar to the genomic unstable type proposed by Sjodahl and colleagues.Class 3 is characterized by a basal-like phenotype and FGFR3 mutation,with a short overall survival, representing the dormant tumor state of NMIBC, and gradually changing to basal-like MIBC with FGFR3 mutation.Some studies have proved that the progress of NMIBC is also affected by tumor differentiation status[7].However,the current NMIBC classification has little research on tumor differentiation status, so more studies are needed to improve the existing classification methods in the future, in order to better assess the risk of NMIBC progression.

2.MIBC molecular classification

MIBC can be divided into different molecular classifications according to different gene expression, and these molecular classifications may be related to clinical characteristics, treatment response, prognosis assessment.[8].Currently, the mainstream molecular classification system believes that bladder cancer can be divided into at least two categories: luminal type and basal type[9-14].The luminal tumors are characterized by high expression of luminal cell markers (such as KRT-20, FGFR3, FOXA1, GATA3),activated PPAR receptors and estrogen receptors, and their prognosis is relatively good.The basal type tumors are characterized by high expression of basal cell markers (e.g.Krt5/6, KRT14) and poor prognosis.UNC classification[9] found a “claudin-low” subtype similar to breast cancer subtype on the basis of basal MIBC, and“Claudin-low” subtype belongs to basal type, but there is no significant difference in disease-specific survival rate and overall survival rate between them.The results of the study also found that the molecular classification of high-grade bladder cancer is highly similar to that of breast cancer, and whether some treatment methods of breast cancer can be applied to bladder cancer needs to be further studied.TCGA classification in 2017[10] further subdivided luminal and basal types into five subtypes: luminal,luminal infiltrating, luminal papillary cell, basal squamous cell and neural type.Luminal cell markers were highly expressed in luminal type, luminal infiltrating type and luminal papillary cell type.Basal squamous cell type showed high expression of basal cell markers.Neurotypes highly express genes for neural differentiation.In 2012,Lund classification[11] classified bladder cancer into Urobasal A type, Urobasal B type, invasive type, squamous cell carcinoma-like type and genomic instability type.In 2018, Lund classification[12]divided bladder cancer into 10 subtypes: Uroa-prog (UroA, UroA)UroB, UroC, genome unstable (GU), genetically unstable infiltrating(GU-INF), interstitial (MES-like), basal/squamous (Ba/Sq), basal/squamous infiltrating (Ba/ SQ-INF), small cell neuroendocrine(MES-like).Uro and GU types were classified as luminal types,while Ba/Sq, Ba/ SQ-INF, MES-like and MES-like were classified as basal types.MAD classification[13] proposed p53-like type on the basis of luminal type.P53 wild-type gene was significantly activated in p53-like type and was resistant to chemotherapy.All resistant MIBCs became p53-like type after chemotherapy, suggesting that P53 gene may play an important role in chemotherapy.NanoString classification[14] proposed a third type based on these two types:double-negative type.Double-negative type does not express luminal or basal type genes, but almost all immune checkpoint ligands are overexpressed in double-negative type tumors, so patients with this type may benefit from immunotherapy.

The above classification methods have been published and are commonly used, but there are still some rare classification methods.For example, in 2022, Zhou Fangjian’s team from the Department of Urology, Sun Yat-sen University Cancer Center[15] believed that most previous molecular classification studies focused on mRNA expression level.Therefore, they analyzed 3764 lncrnas of 320 MIBC patients from TCGA database and divided MIBC into four types: Cluster 1, Cluster 2, Cluster 3 and Cluster 4 (median disease-free survival were 11.8, 15.3, 17.9 and 18.9 months, respectively).Cluster 1 show high frequency of TTN gene mutations and enrichment of M2 macrophages.Cluster 2 show potential sensitivity to immunotherapy and insensitivity to chemotherapy.Cluster3 is potentially sensitive to immunotherapy,but not to chemotherapy.Cluster4 is insensitive to immunotherapy and sensitive to chemotherapy.However, this classification has not yet been applied to the clinic and needs to be confirmed by largescale, multicenter and prospective studies.In 2021, Zhang Yi’s team[16] of the First Affiliated Hospital of Zhengzhou University put forward the idea of immune classification, believing that bladder urothelial carcinoma can be divided into four types: immune ignorance type, immune inactivation type, cold tumor type and hot tumor type.The immunonaive type has the characteristics of immunosuppression while the immunoinactive type has no response to immunotherapy.These two types of neutrophils are relatively increased, and neutrophils have opposite antitumor immunity,so the prognosis of these two types is poor.The cold tumor type has no immune cell infiltration at all, and the pathological type is mostly papillary type.It has early stage and good prognosis.The hot tumor type is infiltrated by T cells and other immune cells, and has good anti-tumor immunity.The prognosis of the hot tumor type is second only to the cold tumor type.In these four different subtypes,different tumor-intrinsic signaling pathways are involved in the infiltration and rejection of different immune cells, so targeting these tumor-intrinsic signaling pathways may be an effective approach for bladder cancer treatment in the future.Both mainstream and nonmainstream classification systems classify bladder cancer from the genetic and molecular levels, which promote our understanding of bladder cancer.However, different classification systems have different methods and different standards, which hinder the practical clinical application to a certain extent.

In 2019, Kamoun et al.[17] proposed consensus molecular classification in order to integrate the current six mainstream MIBC classification systems.Based on the analysis of 1750 MIBC genes,six subtypes were defined: luminal papillary type (LUM-P), luminal nonspecific type (LUM-NS), luminal unstable type (LUM-U), matrix rich type (Sr), basal/squamous type (Ba/Sq), and neuroendocrine type (NE-like).The proportion of young patients with LUM-P type is high, most of them are T2 stage, and FGFR3 mutation is the main mutation, 38% of cases have KDM6A mutation.33% have CDKN2A homozygous deletion, and the prognosis is the best.Some studies have shown that inhibition of mutated FGFR3 can reduce the malignant potential of bladder cancer, confirming that FGFR3 is an effective therapeutic target for bladder cancer[18].There are many elderly patients with LUM-NS type, most of them are in T3-T4 stage, mainly manifested as PPARG(76% of tumors) and ELF3(35%of tumors) mutations, which may be sensitive to neoadjuvant chemotherapy, and its prognosis is similar to that of LUM-P type.Lum-u type is the most unstable subtype of luminal type, mainly with ERCC2 and TP53 mutations, and has a low overall survival rate.Sr type is mainly characterized by the overexpression of smooth muscle, myofibroblasts, fibroblasts and endothelial genes, and its prognosis is second only to that of LUM-P type.Ba/Sq type is more common in female and higher stage tumors, most of which are stage T3-T4.Histological examination shows that 79% of the tumors are squamous differentiated and highly express EGFR receptor and its ligands, suggesting that they may benefit from EGFR targeted therapy.The higher expression of immune checkpoint markers and antigen presentation mechanism genes in Ba/Sq tumors suggests that these tumors may be more sensitive to immunotherapy.At the same time, some studies have shown that patients with Ba/Sq type may benefit from cisplatin-based neoadjuvant chemotherapy[19].Although this subtype has a poor prognosis, there are many treatment options.More clinical trials are needed to analyze the best treatment options for this subtype in the future.Ne-like type is the most rare type, showing elevated cell cycle activity and hypoxic signal, manifested by mutation or deletion of TP53 and RB1, and has the worst prognosis.Further analysis of the consensus molecular subtypes of bladder cancer may provide the most appropriate treatment for each subtype.However, it should be noted that bladder cancer is a heterogeneous tumor, and there may be multiple subtypes in the same tumor, which may affect the precise treatment of molecular classification.Therefore, further studies are needed to improve the accuracy of molecular classification.

3.The guiding significance of molecular classification for treatment

3.1 Molecular classification predicts the efficacy and prognosis of neoadjuvant chemotherapy (NAC)

Chemotherapy has always been the cornerstone of the treatment of bladder cancer patients.For MIBC patients, the National Comprehensive Cancer Network (NCCN) guidelines recommend cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy[20], and NAC is also the first-line treatment for locally advanced or metastatic bladder cancer[21].However, not all bladder cancer patients benefit from NAC.Studies have shown that NAC can only improve the overall survival rate of 30～40% of patients[22], and the clinical application rate of NAC is not high due to its serious toxic and side effects.Therefore, the primary task of clinical research is to identify biomarkers that can be used to predict response to NAC treatment.Seiler et al.confirmed that different molecular classifications have different drug sensitivities to NAC.They found that basal subtypes benefit more from NAC than luminal subtypes, and NE-like subtypes, which have the worst prognosis among all subtypes, can also benefit from NAC[23].A study by Choi et al.showed that p53-like bladder cancer was resistant to NAC[13].McConkey et al.found that patients with Ba/Sq type may benefit from NAC[19].However, Ba/Sq type highly expresses basal cell markers[12], and p53-like type belongs to luminal type[13], thus confirming the research results of Seiler et al.The advantage of molecular classification is that it can select patients who are suitable for NAC; therefore, patients with basal or neuroendocrine bladder cancer should receive NAC first rather than immediate radical cystectomy.

3.2 Molecular classification predicts the efficacy and prognosis of immunotherapy

In the past decades, there has been no significant progress in the treatment of MIBC, and standard treatment is limited to chemotherapy and radical cystectomy[24].The emergence of immune checkpoint inhibitors (ICIs) has brought great changes to the treatment of bladder cancer.ICIs has become the secondline treatment for patients who have lost the opportunity for radical resection and metastatic bladder cancer, and the firstline treatment for patients who are not suitable for platinumbased chemotherapy[20].Programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) inhibitors are the most mature immunotherapy methods.The most widely used ICIs include PD-L1 (atezolizumab, durvalumab, and avelumab) and PD-1(nivolumab and pembrolizumab).However, not all patients can benefit from immunotherapy[25].The development and validation of tumor markers and molecular typing are crucial for patients receiving ICIs.In 2017, TCGA classification showed moderate expression of PD-L1 and CTLA-4 in the lumen infiltration type.Although this subtype was not sensitive to NAC, Atezolizumab showed certain responsiveness to this subtype.Basal/squamous cell types with high expression of immune markers PD-L1 and CTLA4, cisplatin-based NAC and immune checkpoint inhibitors are suitable treatment options[10].Although the neurologic type has the shortest overall survival, it is the most sensitive to atezolizumab[26], so the neurologic type of bladder cancer patients may be the first suitable for immunotherapy.Rosenberg JE et al.found in their study that Cluster I tumors (similar to luminal papillary cell type)in TCGA classification in 2014 were resistant to atezolizumab,while Cluster II tumors (similar to p53-like type/luminal infiltrating type) were most sensitive to atezolizumab[27].P53-like MIBC in MAD classification is significantly resistant to chemotherapy[13],suggesting that immunotherapy should be given priority to patients with p53-like MIBC.With the application of more and more ICIs in clinical practice, it has been found that ICIs combined with chemotherapy, radiotherapy and dual immunotherapy of two ICIs can improve the prognosis of patients more than single ICIs therapy[28].Therefore, the future molecular classification of bladder cancer still needs to predict the drug reactivity and prognosis of multiple combination therapies, in order to provide more detailed and accurate individualized treatment for bladder cancer patients.

3.3 Molecular classification predicts the efficacy and prognosis of targeted therapy

Advanced bladder cancer is a malignant tumor with rapid progression.The emergence of molecular targeted therapy can help optimize first-line and second-line treatment, thus prolonging the overall survival of patients[29].With the development of molecular classification, a variety of molecular markers related to tumorigenesis and development have been proved to be therapeutic targets, among which fibroblast growth factor receptor 3 (FGFR3) is a common tumor driver gene in bladder cancer.Early clinical trials have shown that FGFR3 targeting agents can prolong overall survival of FGFR3 mutant bladder cancer[30-32].Luminal MIBC is characterized by FGFR3 mutations, suggesting that this subtype may benefit from FGFR3-targeted therapy.Studies have found that human epidermal growth factor receptor 2 (HER2) is overexpressed in 40% of bladder cancer specimens, EGFR is overexpressed in 74% of bladder cancer specimens, EGFR is highly expressed in basal MIBC, and HER2 is highly expressed in luminal MIBC[33].The currently used EGFR tyrosine kinase inhibitor gefitinib is less effective for advanced MIBC, while erlotinib, another EGFR tyrosine kinase inhibitor,reduces clinicopathological stage and improves postoperative survival in patients undergoing radical cystectomy.Trastuzumab, a monoclonal antibody against HER2, has achieved certain efficacy in MIBC with HER2 overexpression[29].Although the clinical application of anti-HER2 targeted therapy in bladder cancer is rare,more and more clinical trials have shown that anti-HER2 targeted therapy can provide a new therapeutic strategy for bladder cancer.The molecular classification and treatment of bladder cancer are similar to those of breast cancer, and luminal MIBC highly expresses estrogen receptor (ER).Therefore, can MIBC also be used as a reference for the treatment of breast cancer for endocrine therapy or ER-targeted therapy? Many studies have confirmed the close relationship between ER and bladder cancer.Therefore, in-depth study of the efficacy of ER on bladder cancer will provide new ideas for the treatment of ER+, especially female bladder cancer patients.

4.Challenges and Prospects

The molecular classification system of bladder cancer is constantly updated.However, the current molecular classification system mainly focuses on genomics and transcriptomics.Proteomics,immunoomics and metabolomics are also closely related to the occurrence and development of bladder cancer.Therefore, more omics should be introduced into the molecular classification of bladder cancer.In addition, due to the high heterogeneity of bladder cancer, there may be two or more subtypes in the tumor tissue of the same patient, and the subtypes may change with tumor progression.More studies are needed to improve the molecular classification system in the future.Compared with the traditional pathological classification, molecular classification can more comprehensively analyze the internal characteristics of bladder cancer and provide guidance for individualized treatment of bladder cancer.In the future, molecular classification will become an important supplement to traditional pathological classification.

Description of conflicts of interest and author contribution

Conflicts of interest: The content of this article does not involve relevant conflicts of interest, and the study does not involve any direct or indirect financial or interest sponsorship by any manufacturer or other economic organization.

Author Contribution: Liu Zhitao: Mainly engaged in literature screening, review writing and other work.Liu Fujin: After carefully reviewing the first draft, provide relevant revision suggestions,improve them, and form a revised draft.Wang Fei: The overall control of the article, the article content proofreading and so on.

Supported by: Hainan Clinical Medical Center Construction Project.