Assessment of Molecular Biomarkers for Bladder Cancer Diagnosis, Prognosis and Therapy

Introduction
2. Molecular biology behind bladder cancer
2.1 Genetic Biomarkers
2.1.1. Telomere vs Telomerase
2.1.2. Tumor Suppressor P53
2.1.3. PIK3CA/AKT1 Pathway
2.2 Epigenetic Biomarkers
2.2.1. Twist 1
2.2.2. Nidogen
2.2.3. Vimentin
3. Bladder cancer clinical management
3.1 Diagnosis
3.2 Staging
3.3 Grading
4. Bladder Cancer Classification: Histological Vs Molecular
4.1 Histological classification of bladder cancer
4.2 Molecular classification of bladder cancer
5. Treatment of bladder cancer
5.1 Conventional treatments
5.2 Innovative treatments
5.3 Advances in bladder cancer’s immunotherapy
 Introduction :
Cancer is a complex pathology, characterized by a multitude of molecular and genetic abnormalities,
affecting cell proliferation and differentiation, apoptosis, as well as mobility (invasion). All these
alterations represent potential targets for the development of targeted therapy. These new therapeutics
inhibit cell growth and are called cytostatics in contrast to conventional chemotherapies, known as
cytotoxic. The improvement in understanding the molecular biology of bladder tumors has made it
possible to define the different signaling pathways, involved both in carcinogenesis and in tumor
progression, and to isolate, within these pathways, certain key molecules serving as both prognostic
markers and potential therapeutic targets. Locally advanced and/or metastatic bladder cancer is
characterized by alterations in the p53 and retinoblastoma (Rb) genes, regulators of the cell cycle, which
interact with the Ras-mitogen activated protein kinase (MPAK) transduction pathway. Overexpression
of tyrosine kinase receptors, including EGFR, VEFGR, and HER2/neu, is correlated with tumor
progression, and activation of the phosphatidylinositol-3 kinase (PI-3K) pathway is implicated in tumor
invasion and inhibition of apoptosis. The molecular heterogeneity of bladder tumors will require the
combination of several molecules to provide optimal personalized treatment. Furthermore, modulation
of signaling pathways by these new molecules allows for the restoration of chemosensitivity to cytotoxic
drugs, which can then be associated with targeted treatments.
Inserer schema ici
Figure 2. Évènements moléculaires de l’angiogenèse. L’activation de la voie de transduction EGFR
stimule l’expression de VEGF, des metalloprotéinases (MMPs) et de l’’interleukine 8 (IL-8). Le VEGF
active l’angiogenèse et les MMPs et IL-8 stimule la migration des cellules endothéliales. L’activation du
VEGFR stimule l’activation de l’angiogenèse. Le p53 inhibe l’angiogenèse par augmentation de
l’expression du facteur anti-angiogénique TSP-1 (thrombospondine-1).
Molecular biology behind bladder cancer
Many detailed studies of the molecular pathogenesis of bladder cancer have been published during the
past three decades, identifying important roles for many of the classic cancer pathways in bladder cancer
development.
Two potential pathways of the pathogenesis of papillary non-muscle-invasive bladder cancer and solid
muscle-invasive bladder cancer have been identified (43). Low-grade papillary tumours can arise via
simple hyperplasia and minimal dysplasia, and are characterized at the molecular level by loss of
heterozygosity of chromosome 9 and activating mutations of genes encoding fibroblast growth factor
receptor 3 (FGFR3), telomerase reverse transcriptase (TERT), phosphatidylinositol 4,5-bisphosphate 3-
kinase catalytic subunit alpha isoform (PIK3CA) and inactivating mutations of STAG2 (which encodes
cohesin subunit SA-2). All these genes have roles in cell proliferation, division and growth .
MIBC is thought to arise via flat dysplasia and carcinoma in situ (CIS), which commonly show TP53
mutations in addition to loss of heterozygosity at chromosome 9, but fewer FGFR3 mutations. Low-
grade papillary NMIBCs might progress to MIBCs as a result of CDKN2A loss.
Numerous potential differences in the molecular pathways to the major tumor types and their subtypes
are known (43). Nonetheless, recent large-scale mutational and expression analyses in bladder cancer
made possible by next-generation sequencing and other new techniques have uncovered multiple genes
and pathways important for bladder cancer development, many of which were previously unknown.
Genes involved in cell cycle control, chromatin regulation, and receptor tyrosine and PI 3-kinase-mTOR
signaling pathways are commonly mutated in muscle-invasive bladder cancer, and most cancers have
involvement of more than one of these pathways. In addition, expression-based analyses have enabled
identification of two distinct types of bladder cancer, basal and luminal, that are similar to those same
subsets of breast cancer and have prognostic and therapeutic significance (44).
These observations are leading to several novel therapeutic approaches in bladder cancer, providing
optimism for therapeutic progress shortly (44). However, despite the extensive molecular
characterization of bladder cancer, no effective targeted therapy has yet made it into clinical routine.
Genetic alterations
Since the discovery of HRAS as the first oncogene in bladder cancer, multiple genes commonly subject
to a mutation in bladder cancer have been identified, including TP53, RB1, TSC1, FGFR3, PIK3CA,
and TERT. Furthermore, comparative genomic hybridization and related techniques were used
extensively in bladder cancer, leading to the identification of multiple amplified and deleted genes,
including PPARG, E2F3, EGFR, CCND1, and MDM2 which are amplified, and CDKN2A and RB1
which are commonly deleted (44–50).
Bladder cancer displays a wide range of frequently occurring somatic DNA alterations, including both
mutations and copy number alterations. The majority of recurring somatic mutations occur in genes
involved in mitogenic signaling, cell cycle regulatory pathways, or chromatin modification. The main
mitogenic pathways affected include the PI3K/AKT/mTOR pathway with activating mutations in
PIK3CA, PIK3R1, PIK3R2, MTOR and AKT1, inactivating mutations in pathway regulators such as
NF1, PTEN, TSC1 and TSC2 and telomere maintain through TERT promoter reactivating mutations
(51,52). The overall most common mutation target is the promoter region of the TERT gene, seen in up
to 70% of bladder cancers (53). These mutations lead to increased TERT expression and result in
avoiding cell senescence and maintaining telomere ends (54).

Figure

Pathways are indicated by solid arrows; broken arrows indicate uncertain relationships. Relative
frequencies of predominant molecular alterations are provided as summarized percentages. A third
hypothetical pathway to the development of high-grade papillary tumors is shown. The possible pathway
leading to T1 disease is provided implicating 2 different T1 entities.
In addition to mutations, copy number alterations play an important role in bladder cancer
tumorigenesis. Watters et al. showed that the majority of bladder carcinomas are polysomic for
chromosomes 8 and 11. These characteristics were reflected by the high copy number variation (CNV)
detected in MYC and CCND1. CCND1 regulates the cell cycle transition from G1 to S phase by the
cyclin-dependent kinases CDK4 and CDK6. Its overexpression results in a dysregulated CDK activity
followed by rapid cell growth. Amplifications of both genes have been reported to be associated with
muscle-invasive BC (56).

CNV was found also in ERBB2 in the DNA of primary tumor and lymph node metastasis. ERBB2
regulates epithelial-mesenchymal transition (EMT), migration, and tumor invasion by modulating
extracellular matrix (ECM) components. CNV is present in various genes of genomic DNA derived
from primary tumors, lymph node metastasis, and cfDNA from serum in BC patients treated with radical
cystectomy. CNV in specific genes of genomic DNA derived from the primary tumor, lymph node
metastasis, and cfDNA is associated with unfavorable clinicopathologic BC features. In addition,
patients with CNV in ERBB2 and TOP2A in the primary tumor are at increased risk for disease
recurrence (57).
CNVs of CEP63, FOSL2, and PAQR6 with high aberration frequencies (67.7%, 56.9%, and 60.0%,
respectively) were found in bladder cancer tumors. These CNVs were correlated with advanced tumor
stage and high grade. Moreover, it was revealed that CNVs of CEP63 and FOSL2 were independent
prognostic factors for disease-free survival (DFS) of non-muscle-invasive bladder cancer patients, while
CNVs of FOSL2 and PAQR6 were independent prognostic factors for DFS of muscle-invasive bladder
cancer patients.
Copy number variations in CEP63, FOSL2, and PAQR6 were capable of predicting DFS and may serve
as promising signatures for the prognosis of bladder cancer (58).
Epigenetic alterations

In the past decade, DNA methylation analysis has been revolutionized by two technological advances:
bisulfite modification of DNA and methylation-specific polymerase chain reaction (MSP) (59). Major
advances and improvements in high-throughput genomic sequencing have elevated the understanding of
the mutational landscape and remarkable heterogeneity in bladder cancers, which are related in part to
grade and histology (41). As a result, our ability to study the epigenome has improved, adding to our
understanding of how epigenetic dysregulation contributes to the pathogenesis of urothelial carcinoma
(60).
DNA methylation has been the most studied epigenetic alteration in bladder cancer. DNA methylation
occurs when a methyl group forms a covalent bond to the 5′ position of a cytosine ring (61). The
methylation profile can distinguish tumor types, subtypes, and perhaps the response to chemotherapeutic
agents and survival. Methylation changes often precede apparent malignant changes and thus may be of
use in early diagnosis of cancer. Moreover, sensitive detection of cancer cells could be obtained from
different body fluids in many cancers (59).
Substantial evidence shows that the epigenome of BC cells displays profound alterations in DNA
methylation, histone modifications, and nucleosome positioning. In this context, a few well-known
tumor suppressor genes, including CDH1, CDH13, RASSF1A, MLH1, and DAPK, have been reported
to be frequently hypermethylated and silenced in BC, resulting in deregulated cell proliferation(62).
In addition to global hypomethylation of repetitive elements, such as long interspersed nuclear elements
(LINE-1), It has been shown that a specific LINE-1 located within the mesenchymal-epithelial transition
factor oncogene (L1-MET) is hypomethylated and transcriptionally active in BC, accompanied by the
presence of a nucleosome-depleted region just upstream of the transcription start site, active histone
marks, and the histone variant H2A.Z (63).
In bladder cancer, the two mutually exclusive molecular pathways for the formation of noninvasive and
invasive tumors also differ epigenetically in addition to genetically. A genome-wide analysis of DNA
methylation patterns in noninvasive and invasive urothelial tumors revealed a distinct hypomethylation
pattern only in noninvasive tumors and widespread hypermethylation in invasive tumors, suggesting that
they arise via distinct epigenetic pathways (64).
Furthermore, distinct methylation patterns have been identified between non-muscle-invasive bladder
cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), as well as between FGFR3-mutant and
wild-type tumors. Given these distinctions in expression, methylated genes have been proposed as
diagnostic and prognostic biomarkers for patients with bladder cancer. Indeed, several studies have
revealed that methylated genes, including CDH1, FHIT, LAMC2, RASSF1A, TIMP3, SFRP1, SOX9,
PMF1, and RUNX3 are associated with poor survival in patients with MIBC (65).

Bladder cancer clinical management

Diagnosis

There is evidence that the patient experience for people with bladder cancer is
worse than people with other cancer (66). The involvement of the urogenital
tract and the nature of the treatments give this cancer a strong psychological
impact, in addition to the physical impact of the disease and its treatments,
which is often profound. The prevalence of the condition and the nature of its
management make bladder cancer one of the most expensive cancers due to
surveillance and treatment of recurrences (67).

The most common presentation of bladder cancer is visible, or gross,

hematuria, but patients can also present with isolated microscopic hematuria
(urinalysis showing ≥3 red blood cells per high-power field), irritative voiding
symptoms, or a tumor incidentally discovered on imaging. The risk of bladder
cancer is approximately 4% in patients with microscopic hematuria and 16.5%
in those with gross hematuria (68). The diagnosis is often delayed due to the
similarity of these symptoms to benign disorders (e.g., urinary tract infection,
interstitial cystitis, prostatitis, passage of renal calculi), and delays can lead to a
worsened prognosis due to more advanced stage at diagnosis (69). Patients
with macroscopic hematuria or suspected of having bladder cancer require
bladder wall exploration by cystoscopy (70). The examination frequently also
includes other tests such as urine testing and cytology to detect infections and
atypical cells (71).

Although urine cytology is easy to obtain and highly sensitive and specific for
high-grade lesions, it has a low sensitivity for low-grade lesions and a high rate
of equivocal results. Because of the low sensitivity for small lesions, urine
cytology should be used as an adjunct test to aid in the diagnosis of bladder
cancer (72).
Urine cytology has a sensitivity of 25%, specificity of 91%, high positive
predictive value but low negative predictive value (68). Ultrasound is excellent
in determining whether a mass is cystic or not and whether a cystic lesion is a
simple cyst or minimally complicated or complicated. Lesions that are not
simple cysts require further work-up with CT or MRI. CT urography
outperforms ultrasound and radiography in the evaluation of renal masses and
urinary tract calculi with sensitivity and specificity for upper tract urothelial
malignancies of 67-100% and 93-99%, respectively (73).
Rates of bladder cancer detected during cystoscopic evaluation for microscopic hematuria vary
widely based on the populations under investigation and criteria used for significant hematuria
(70). If a tumor lesion is found in the bladder, the first step of the initial management consists of
a transurethral resection of the bladder tumor (TURBT). The TURBT is both a therapeutic and a
diagnostic procedure, where successful removal of the entire lesion may be curative if the tumor
is not invasive (74).

Staging

The pathological evaluation is performed on the resected TURBT sample to determine the depth
of invasion. This process is called staging. The stage of cancer describes the extent (amount) of
cancer in the body. It helps determine how serious the cancer is and how best to treat it. A
staging system is a standard way for the cancer care team to describe how far a cancer has
spread. The staging system most often used for bladder cancer is the American Joint Committee
on Cancer TNM system, which is based on 3 key pieces of information (Table)

 T describes how far the main (primary) tumor has grown through the bladder wall and
whether it has grown into nearby tissues.

 N indicates any cancer spread to lymph nodes near the bladder. Lymph nodes are bean-
sized collections of immune system cells, to which cancers often spread first.

 M indicates if the cancer has spread (metastasized) to distant sites, such as other organs,
like the lungs or liver, or lymph nodes that are not near the bladder (75).

Grading

Tumor grade is a critical parameter in determining the management approach for patients with
non-muscle-invasive bladder cancer. In 1998 a consensus conference convened by the
International Society of Urological Pathology (ISUP) proposed a novel system to replace the
World Health Organisation (WHO) 1973 grading system (76) and this recommendation was
incorporated into the WHO classification of bladder tumors in 2004 (Figure 8).

The WHO 2004 grading system has resulted in a significant increase in high-risk category
tumors with a consequent risk of overtreatment. This is important as BCG therapy, that serves as
first line treatment for NMIBC, can have significant side effects, while in rare cases a patient
may even undergo cystectomy for intractable BCG symptoms, which would be a tragedy if the
Table. Tumor, Node, Metastasis (TNM) classification system for bladder cancer

Stage Stage grouping Stage description

Ta The cancer is a non-invasive papillary carcinoma (Ta). It has grown toward the
0a N0 hollow center of the bladder but has not grown into the connective tissue or
M0 muscle of the bladder wall.
It has not spread to nearby lymph nodes (N0) or distant sites (M0).
The cancer is a flat, non-invasive carcinoma (Tis), also known as flat
Tis
carcinoma in situ (CIS). The cancer is growing in the inner lining layer of the
0is
N0 bladder only. It has not grown inward toward the
hollow part of the bladder, nor has it invaded the connective tissue or muscle
M0
of the bladder wall. It has not spread to nearby lymph nodes (N0) or distant
sites (M0).
T1 The cancer has grown into the layer of connective tissue under the lining layer
N0 of the bladder, but has not reached the layer of muscle in the bladder wall (T1).
I

The cancer has grown through the muscle layer of the bladder and into the
T3a, T3b layer of fatty tissue that surrounds the bladder (T3a or T3b).
or It might have spread into the prostate, seminal vesicles, uterus, or vagina, but
T4a it's not growing into the pelvic or abdominal wall (T4a).
N0 The cancer has not spread to nearby lymph nodes (N0) or to distant sites (M0).
IIIA M0
OR
The cancer has:
• grown into the layer of connective tissue under the lining of the
bladder wall (T1), OR
• into the muscle layer of the bladder wall (T2), OR
• into the layer of fatty tissue that surrounds the bladder, (T3a or T3b)
OR
T1-4a • it might have spread into the prostate, seminal vesicles, uterus, or
N1 vagina, but it's not
M0 growing into the pelvic or abdominal wall (T4a).
AND the cancer has spread to 1 nearby lymph node in the true pelvis (N1). It
has not spread to distant sites (M0).
The cancer has:
 grown into the layer of connective tissue under the lining of the
bladder wall (T1), OR
T1-T4a  into the muscle layer of the bladder wall (T2), OR
IIIB
N2 or N3 M0  into the layer of fatty tissue that surrounds the bladder (T3a or T3b),
OR
 it might have spread into the prostate, seminal vesicles, uterus,
or vagina, but it's not growing into the pelvic or abdominal wall
(T4a).
AND the cancer has spread to 2 or more lymph nodes in the true pelvis (N2) or
to lymph nodes along the common iliac arteries (N3).
It has not spread to distant sites (M0).
T4b Any N The cancer has grown through the bladder wall into the pelvic or abdominal
M0 wall (T4b).

Any T Any It might or might not have spread to nearby

N M1a lymph nodes (Any N). It has not spread to
IVA distant sites (M0).

OR

The cancer might or might not have grown through the wall of the bladder into
nearby organs (Any T).
It might or might not have spread to nearby
 lymph nodes (Any N). It has spread to distant
lymph nodes (M1a).
The cancer might or might not have grown through the wall of the bladder into
Any T Any
nearby organs (Any T).
IVB N M1b
It might or might not have spread to nearby lymph nodes (Any N).
It has spread to 1 or more distant organs, such as the bones, liver, or lungs
(M1b).

1. Bladder Cancer Classification: Histological Vs Molecular

1.1 Histological classification of bladder cancer

The stage classification differentiates between non-muscle invasive (NMI; Tis, Ta, and T1) and
muscle-invasive tumors (T2, T3, and T4) according to the invasion depth. Ta tumors are
restricted to the urothelium; T1 tumors have invaded the lamina propria; and T2, T3, and T4
tumors have invaded the superficial muscle, perivesical fat, and surrounding organs,
respectively. Tis is poorly understood and believed to be a precursor of muscle-invasive tumors.
The majority of patients, 70%, initially present with NMI tumors, however, up to 70% of these
develop local recurrences, and patients may have several recurrences (Figure 9) (79).

Until 2016 different bladder tumor grading systems have been introduced, with generally good
acceptance. One of the most widely accepted systems has been the WHO 1973 system, which is
still used in some parts of Europe (80). However, the currently recommended system is the new
WHO 2016 Grading system, which is being proposed for universal use and should be adopted
world-wide (81).

Urothelial cell carcinoma patients are stratified by pathologic stage and grade, the basis of
clinical decision-making. A large study carried by Ooms et al., showed that 50% of pathologists
graded differently the same tumors and that the same tumor was graded differently at different
times by the same pathologist, stating that inter- and intraindividual inconsistency in the grading
of bladder tumors might invalidate the usefulness of histologic grading in clinical decision
making (82).
 Therefore, to improve the accuracy of grading the WHO adopted a new classification system for
grade in 2004 (83). The major difference is in the classification of papillary tumours, which are
classified as grades 1, 2 and 3 in the older system and as papillary urothelial malignancy of low
malignant potential (PUNLMP; equivalent to grade 1), low-grade papillary urothelial carcinoma
or high-grade papillary urothelial carcinoma in the WHO/ISUP 2004 classification (84).
However, predicting recurrence, progression or response to treatment continue to pose a real
challenge to pathologists.

Figure 9: Bladder cancer WHO 1973 and 2004 Staging of urothelial tumors (30)

a) Staging of bladder cancer according to the Tumour-Node-Metastasis (TNM) system

b) Grading according to the 1973 World Health Organization (WHO) and 2004 WHO/ International
Society of Urological Pathology (ISUP).

1.2 Molecular classification of bladder cancer

Since bladder cancers are biologically heterogeneous and have widely variable patterns, many
groups have directed their research into finding a novel classification based on molecular
signature that will permit better understanding and management of the disease. Recently, a
molecular taxonomy for urothelial carcinoma based on integrated genomics using gene
expression profiles could define five major urothelial carcinoma subtypes: urobasal A,
genomically unstable, urobasal B, squamous cell carcinoma like, and an infiltrated class of
tumors. The subtypes show distinct clinical outcomes suggesting that the molecular phenotypes
can be an intrinsic property of the tumors (Table 2) (85). Also, in 2013 Damrauer et al., have
demonstrated that there are at least two molecularly and clinically distinct subtypes of high-grade
bladder cancer termed “luminal” and “basal-like,” which have characteristics of different stages
of urothelial differentiation, reflecting the luminal and basal-like molecular subtypes of breast
cancer, with clinically meaningful differences in outcome (86).

The whole genome mRNA expression profiling and unsupervised hierarchical cluster analyses
carried on three data cohorts (Chungbuk, Lund and UCSF) and a discovery cohort of 73 tumors
identified three molecular subtypes of MIBC that resembles established molecular subtypes of
breast cancer: luminal, basal and P53-like MIBC which was consistently resistant to neoadjuvant
chemotherapy (87). In 2017 Sjodahl et al., analyzed 307 advanced bladder cancers both by
genome gene expression analysis and by immunohistochemistry to explore 28 proteins and
describe five tumor‐cell phenotypes of advanced urothelial carcinoma: urothelial‐like,
genomically unstable, basal/SCC-like, mesenchymal-like, and small-cell/neuroendocrine‐like,
and showed that cancers with different tumor‐cell phenotypes may converge to the global mRNA
analyses, while cases with identical tumor‐cell phenotypes may diverge (88).

Recently, a Meta-cohort Analysis of 2411 unique tumors encompassing NMIBC and MIBC
tumors was analyzed, based on gene expression. The analysis revealed six molecular subtypes
with different overall survival and molecular features. They have also shown that NMIBC, with a
high risk of progression, displays the molecular features of MIBC (89). Several other groups
also sought to define the molecular subtypes in BC (90). These studies have based their finding
on genetic, epigenetic, transcriptomic and proteomic investigations and the observed differences
in results in the established subtypes are due to differences in study targets, methods and cohort
sizes (Figure 10).

The rapid progress of genomic characterization of UC in recent years has resulted in a variety of
subtypes taxonomies. Although a consensus meeting made a first step towards a standardized
taxonomy, further harmonization will be required in the future for a better understanding and
clinical applicability (95). However, all studies confirm the need of biomarkers that can be used
to orientate patient treatment and avoid unnecessary toxicities in those who resist.
Table 2: Urothelial carcinoma molecular subtyping classifications (36)

UNC (91) Lund Institute (79) MD Anderson TCGA (93) Broad Institute (94) Genomic characteristics
(92)

Basal Squamous-cell like basal cluster 3 basal RB1mutations. NFE2L2mutations.

p16 deletions. Activating
FGFR3mutations. High PD-L1
Urobasal B cluster 4
expression. CDKN2A deletion

Luminal Unfiltrated p53-like cluster 2 immune undifferentiated

Luminal immune

Genomically luminal cluster 1 luminal P53,RB1 deletions.

unstable PPARG,GATA3,ERBB2,E2F3/SOX4
amplifications. ERCC2 alterations.

Urobasal A basal PIK3CA,NFE2L2,ERBB2,ERBB3

mutations.
ActivatingFGFR3mutations. KDM6A
alterations

Figure 10: Overview on current taxonomy on molecular subtypes of urothelial bladder cancer and
expected response to chemotherapy

MDA, the MD Anderson Cancer Center; UNC, the University of North Carolina; TCGA, The Cancer Genome
Atlas; SCC, squamous cell carcinoma.
It is primordial to consider the molecular classification in parallel to histology in decision
making. The routine immunohistochemical detection of PD-L1 presents multiple
unresolved/controversial issues. First, the cut-off score for determining staining positivity is still
a subject of debate. Depending on the cancer subtype, the anti-PD-1/PD-L1 drug to be
administered and the PD-L1 assay used. The cut-off might vary from 1% to 50% positive cells.
Although several pooled analyses showed that, when a cut-off of 1% was used, patients bearing a
PD-L1-positive cancer had a better overall response rate to anti-PD-L1 monoclonal antibodies
compared to their counterparts with PD-L1-negative tumors, the odds ratio was relatively modest
(<2.5). These findings and others correlating PD-L1 expression with the efficacy of treatment
argue for increasing the cut-off value. Moreover, the different detection antibodies, variability in
tissue processing and the absence of standardization regarding the scoring methods used to
quantify PD-L1 expression (the tumor proportion score versus the combined positive score) also
stand in the way of a reproducible assay. Whereas the tumor proportion score only evaluates PD-
L1 expression on cancer cells, the combined positive score takes into account both immune and
tumor PD-L1-positive cells. This latter distinction between the cell types exhibiting membrane
PD-L1 expression is of importance, especially as a stronger correlation was reported between
treatment response and PD-L1 expression on immune cells than that on tumor cells (96).

2. Treatment of bladder cancer

The highest burden of bladder cancer is currently falling on most developed communities across
the globe. But with an anticipated shift in world demographics with growing and aging
populations, mainly on the African continent. An important shifts in exposure to different risk
factors across the world is likely to change over the next decades (97).

Within 5 years, from 50% to 70% of patients diagnosed with NMIBC will recur and 10% to 30%
of patients with NMIBC will progress towards MIBC or metastatic disease despite treatments
(98). When considering a patient's prognosis with NMIBC, it is necessary to consider not only
clinical and pathological factors but also take into account the potential effect of the intravesical
treatment received and molecular alterations present in the tumors (98).

In patients with low-grade Ta disease, the 15-year progression-free survival is 95% with no
cancer-specific mortality. Patients with high-grade Ta tumors have a progression-free survival
(PFS) of 61% and a disease-specific survival (DSS) of 74%, whereas patients with T1 disease
have a PFS of 44% and a DSS of 62%, providing support to the view that grouping all patients
with NMIBC as one disease is misleading as one patient's prognosis can be quite different from
that of another patient (99).

2.1 Conventional treatments

For non-muscle-invasive bladder cancer, the mainstay of treatment is complete resection of the
tumor followed by induction and maintenance immunotherapy with intravesical BCG vaccine or
intravesical chemotherapy. For muscle-invasive bladder cancer, multimodal treatment involving
radical cystectomy with neoadjuvant chemotherapy offers the best chance for cure. Selected
patients with muscle-invasive tumors can be offered bladder-sparing trimodality treatment
consisting of transurethral resection with chemoradiation. Advanced disease is best treated with
systemic cisplatin-based chemotherapy. Immunotherapy is emerging as a viable salvage
treatment for patients in whom first-line chemotherapy cannot control the disease (100).

2.2 Innovative treatments

Due to the genetic instability of bladder cancer, together with individual needs of patients, the
search for different therapy methods is ongoing. The discovery and clinical implementation of
immune-checkpoint inhibitors (ICIs) targeting CTLA4, PD-1 and PD-L1 has revolutionized the
treatment of cancer, as recognized by the 2018 Nobel Prize for Medicine and Physiology. This
groundbreaking new approach has improved the outcomes of patients with various forms of
advanced-stage cancer. However, the majority of patients receiving these therapies, even in
combination, do not derive clinical benefit (101). In fact, it has been shown that high PD-L1
expression is associated with higher stage and grade, higher recurrence, poor survival and worse
outcome in bladder cancer (102,103). Yet, since patients with tumors expressing low PD-L1
might respond to checkpoint inhibition, while inversely, patients with high PD-L1 do not
respond, the need to understand better and enhance the body’s response to ICIs is urgent (104).

Advanced BC, comprised of locally advanced BC and mBC, is generally considered an incurable
disease. To date, cisplatin-based chemotherapy remains the first-line treatment standard for
advanced BC. Carboplatin-based regimens have been substituted in patients for whom the risks
of cisplatin-based chemotherapy are believed to outweigh the potential benefits. Second-line
treatment options for advanced BC have historically been limited (105).

Given the high-risk non-muscle invasive bladder cancer and the negative patient outcome
affected by lack of response to BCG treatment and the lack of response to cisplatin-based
neoadjuvant chemotherapy and cisplatin in muscle-invasive bladder cancer (MIBC) patients, and
effective treatment strategy is of need to improve patients therapy and prognosis (106). A better
understanding of the biology of UC has led to several treatment advances over the last 5 years,
including ICIs, fibroblast growth factor receptor (FGFR) inhibitors, and antibody-drug
conjugates (107).

2.3 Advances in bladder cancer’s immunotherapy

There were 9 US Food and Drug Administration approvals of novel therapies from 2016 to 2019.
Atezolizumab, nivolumab, avelumab, durvalumab, and pembrolizumab were approved for
patients who progressed on platinum-based chemotherapy (CT) in the advanced setting or within
12 months of neoadjuvant or adjuvant (NA/A) treatment (Figure 11) (107).

Atezolizumab and pembrolizumab are approved for treatment-naive, cisplatin-ineligible patients

who have tumors with high PD-L1 expression, as defined below. Erdatifinib, a fibroblast growth
factor receptor (FGFR) tyrosine kinase inhibitor (TKI), was approved for patients who have
tumors with FGFR2 or FGFR3 alterations and progressed on platinum-based CT. Enfortumab
vedotin, an antibody-drug conjugate (ADC), is approved for patients who progressed on
platinum-based CT. High PD-L1 expression is defined as a combined positive score ≥10% for
pembrolizumab and PD-L1 ≥5% for atezolizumab (107). Additionally, CTLA-4 inhibitor
ipilimumab have been approved as most eminent ICIs to treat a myriad of cancers (Figure 11)
(108).

3. PD1/PD-Ls based immunotherapy

Cancer immunotherapy attempts to harness the power and specificity of the immune system to
treat tumors. The molecular identification of human cancer-specific antigens has allowed the
development of antigen-specific immunotherapy (Figure 12) (109).
Figure 11: Timeline of Novel Therapy Approvals for Advanced Urothelial Carcinoma (107)

Figure 12: The FDA-approved immune checkpoint inhibitors (108)

Immune checkpoint inhibitors have revolutionized therapies in various solid tumors. Despite,
promising and durable response rates compared to non-immune therapies, not all patients
respond as effectively to ICIs.

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 (B7-H1, CD274) have attracted
wide attention from researchers in the field of immunotherapy. PD1 is a cell surface receptor that
functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding
of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling
pathways and inhibits T cell activation (110).

PD-L1 and PD-L2 (B7-DC, CD273) belonging to the protein B7 family, are the ligands of PD-1
(111,112). Although a role of PD-L1 in the suppression of anti-tumor immunity and its value as a
predictive biomarker has been suggested by various preclinical and clinical studies, the precise
mechanisms how PD-L2, the other ligand of PD-1, regulates anti-tumor immunity in the tumor
microenvironment are yet to be fully explored (113).

4. Anti-PD1/PD-L1
PD-1/PD-L1 have been shown to exist in many types of cells in addition to T lymphocytes, and
studies have accordingly extended from their suppressive effect on T cell activation and function
to examining their role in other cells (114). Tumor PD-L1 may facilitate localized stage-
advancement of UC and attenuate responses to BCG immunotherapy by neutralizing T cells that
normally guard against cancer invasion from the epithelium into the bladder musculature (115).

A fundamental challenge facing researchers and clinicians is that cancers are inherently robust
biological systems, able to survive, adapt and proliferate despite the perturbations resulting from
anticancer drugs (116). A number of immune checkpoint inhibitors have been approved as first-
line therapy in case of cisplatin-ineligible patients or as second-line therapy for patients with
metastatic urothelial carcinoma of the bladder. About 30% of patients with mUC will respond to
ICIs immunotherapy. Programmed death-ligand expression detected by immunohistochemistry
seems to predict response to immune checkpoint inhibitors in patients with mUC as supported by
the objective response rate (ORR) and overall survival (OS) associated with the response
observed in most clinical trials. Pembrolizumab, an anti-PD-1 antibody, demonstrated better OS
respective to chemotherapy in a randomized phase 3 study for second-line treatment of mUC.
Nivolumab, an aniti-PD-1 antibody, also demonstrated an OS benefit when compared to
controls. Atezolizumab, durvalumab, and avelumab antibodies targeting PD-L1 have also
received approval as second-line treatments for mUC with durable response for more than 1 year
in selected patients. Atezolizumab and pembrolizumab also received approval for first-line
treatment of patients that are ineligible for cisplatin.

A focus on the utility of ICIs in the adjuvant or neoadjuvant setting, or as combination with
chemotherapy, is the basis of some ongoing trials. The identification of a clinically useful
biomarker, single or in association, to determine the optimal ICIs treatment for patients with
mUC is very much needed as emphasized by the current literature (117).

4.1 Anti-PD-L2

PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity
discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has
been assumed to fit snuggly into a pocket on the PD-1 surface (118). Clinical response to
pembrolizumab in patients with head and neck squamous cell carcinoma may be related partly to
blockade of PD-1/PD-L2 interactions (119). In animal models, it was shown that PD-L2
expression alone or simultaneously expressed with PD-L1 in tumor cells significantly suppressed
antitumor immune responses, such as tumor antigen-specific CD8 + T cells, and was involved in
the resistance to treatment with anti-PD-L1 mAb alone. This resistance could be tamed by anti-
PD-1 mAb or combined treatment with anti-PD-L2 mAb (120).

In clinical settings, antitumor immune responses were significantly correlated with PD-L2
expression in the tumor microenvironment in renal cell carcinoma (RCC) and lung squamous
cell carcinoma (LUSC), suggesting that PD-L2 as well as PD-L1 play important roles in evading
antitumor immunity, and that PD-1/PD-L2 blockade must be considered for optimal
immunotherapy in PD-L2-expressing cancers, such as RCC and LUSC (120).

In bladder cancer, a high expression of PD-L2 is detected. This high expression of PD-L2 may
indicate worse prognosis since it was associated with both shorter overall survival and shorter
disease-free survival. PD-L2 may be a potential immunotherapeutic target of immune checkpoint
blockade for bladder cancer (121).
 4.2 Combination therapies with ICIs

Immune checkpoint inhibitors therapy has become a promising therapeutic strategy with
encouraging therapeutic outcomes due to their durable anti-tumor effects. Though, tumor
inherent or acquired resistance to ICIs accompanied with treatment-related toxicities hamper
their clinical utility. Overall, about 60–70% of patients (e.g., melanoma and lung cancer) who
received ICIs show no objective response to intervention, and only 15–25% of patients with
advanced bladder cancer respond to therapy, and even those who initially respond may later
develop acquired resistance (108,122). The resistance to ICIs mainly caused by alterations in the
tumor microenvironment (TME), which in turn, supports angiogenesis and also blocks immune
cell antitumor activities, facilitating tumor cells' evasion from host immunosurveillance (108).
Therefore, for progress to occur, attention has to be directed to the immunobiology of urothelial
cancer, and therapies need to be adjusted (123).

To circumvent the low efficiency of ICIs, treatment combinations and other treatment methods
has been considered for clinical benefit. At present, a number of clinical trials of combined
immunotherapy are ongoing or have reached their endpoints. Clinical trials can provide evidence
for follow-up clinical application, the purpose of which would be to provide more clinical
treatment options and more possibilities for improved overall patient treatment benefit. (124).

The use of ICIs in combination therapy is an emerging trend in tumor immunology. Recent
studies indicate that combination ICI immunotherapy was associated with significantly improved
PFS, OS and ORR in comparison with ICI monotherapy alone. However, most combinations will
induce higher toxicity. Thus, the value of combination immunotherapy remains controversial,
because of the toxic effects induced by combination. The added benefit of each additional drug
has not been assessed against the added toxicity (125).

Results of clinical trials have suggested that the combination of ICIs with chemotherapy, anti-
vascular targeted therapy or anti-HER-2 targeted therapy, and dual ICIs, may improve clinical
treatment efficiency. Clinical trials combining ICIs and radiotherapy for the treatment of
advanced gastric cancer are also ongoing (124).

In bladder cancer, novel combination therapies (Table 3) with chemotherapy, radiations or

targeted drugs were set to improve patients’ response and reduce the emergence of resistance in
the management of the disease (126).

Table 3: Ongoing clinical trials of novel combination therapies in bladder cancer and urothelial cancer (126)

Study number Eligibility Phase Intervention

NCT01928394 Advanced or metastatic bladder cancer Phase I/II Nivolumab and Ipilimumab
1.
NCT03084471 Advanced bladder cancer Phase III Durvalumab and Tremelimumab

Metastatic or advanced transitional cell

NCT03219775 Phase II Nivolumab and Ipilimumab
carcinoma

Nivolumab, Ipilimumab,
NCT03036098 Unresectable or metastatic UC Phase III
Gemcitabine/Cisplatin/Carboplatin

NCT02516241 Unresectable stage IV UC Phase III Durvalumab and Tremelimumab

NCT04223856 Advanced or metastatic UC Phase III EV and Pembrolizumab

NCT03519256 BCG unresponsive high-risk NMIBC Phase II Nivolumab, BMS-986205 and BCG

NCT02560636 Advanced bladder cancer Phase I Pembrolizumab and radiotherapy

Tremelimumab, Durvalumab and

NCT02643303 Bladder cancer Phase I/II
polyICLC

NCT03473743 Metastatic or advanced UC Phase Ib-II Erdafitinib, Cetrelimab and Platinum

NCT03473756 UC Phase Ib/II Rogaratinib and Atezolizumba

NCT04172675 High-risk NMIBC Phase II Erdafitinib, Gemcitabine/Mitomycin C

NCT03745911 Metastatic UC Phase II Paclitaxel and TAK-228

Durvalumab, Olaparib, AZD1775 and

NCT02546661 MIBC Phase I
Vistusertib

NCT03022825 BCG unresponsive high grade NMIBC Phase II ALT-803 and BCG
Telomerase
1.1 Structure and role

The telomerase holoenzyme, which has a highly conserved role in maintaining telomere length,
has long been regarded as a high-profile target in cancer therapy due to the high dependency of
the majority of cancer cells on constitutive and elevated telomerase activity for sustained
proliferation and immortality (127). Telomerase is a DNA polymerase that extends the 3' ends of
chromosomes by processively synthesizing multiple telomeric repeats. It is a unique
ribonucleoprotein (RNP) containing a specialized telomerase reverse transcriptase (TERT) and
telomerase RNA (TER) with its own template and other elements required with TERT for activity
(catalytic core), as well as species-specific TER-binding proteins important for biogenesis and
assembly (core RNP); other proteins bind telomerase transiently or constitutively to allow
association of telomerase and other proteins with telomere ends for regulation of DNA synthesis
(128). Telomerase activity is dependent on the expression of two main core component genes:
TERT encoding the catalytic component and TERC which encodes the RNA component (Figure
13) (129).

Figure 13: Illustration of telomerase components

Human telomeres are composed of complexes between telomeric DNA and a six-protein
complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity
and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex
coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome
ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted
degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a
network of constitutive and context-specific interactions (130).

1.2 TERT and cancers

Scientific evidence have shown that telomerase activity is found in ~90% of human carcinomas
and of particular interest, ~99% of UC of the BCs exhibited a high telomerase activity (Figure
14) (131), conferring to cells replicative immortality via the maintain of their telomere length
stable and reactivating the telomerase. Telomerase is active in stem cells, but in developing
tissues, levels of telomerase activity decrease as progenitor cells cease dividing and begin
differentiating into the cell types that carry out the functions of the tissue (132).

Telomerase activity is maintained somatically in many animals, including the mouse, but is
downregulated during human development and cellular differentiation through transcriptional
repression of TERT, which encodes the catalytic subunit (131).

In human adult non germline tissues, TERT expression and telomerase activity are restricted to
the stem or progenitor cell compartment of some tissues and can be activated in some cell types,
such as lymphocytes, following proliferative stimulation. In the absence of sufficient telomerase
activity, telomeres shorten in proliferating cells during ageing (131).

Over 80% of tumors adopt various regulatory strategies, known as telomere maintenance
mechanisms. They maintain telomere length by reactivating telomerase, and therefore are known
as TERT canonical functions. Approximately 10–15% of tumor cells acquire immortality through
a telomerase-independent mechanism. While telomere lengthening is considered a major
function of telomerase, it can also modulate expression of various genes, such as nuclear factor
NF-κB and Wnt/β-catenin signaling pathway genes (133).
 Figure 14: TERT mutations frequency in multiples cancers (134)

1.3 TERT reactivation mechanisms

The mechanisms inducing TERT reactivation in cancer cells are numerous and diverse. TERT is
subjected to transcriptional, post-transcriptional and epigenetic regulation, but the precise
mechanism of how telomerase is re-activated in cancer cells is poorly understood (135).

1.3.1 Genetic induction

Newly described non-coding mutations in the TERT promoter region of many cancer cells in two
key positions, C250T and C228T, have added another layer of complexity to telomerase
reactivation (Figure 15). These mutations create novel consensus sequences for transcription
factors which can enhance TERT expression. Point mutations in the TERT promoter are highly
recurrent in cancer cases including glioblastoma, melanoma, urothelial bladder, hepatocellular
carcinoma, and thyroid cancers. These mutations generally create new consensus motifs for
transcriptional regulators like ETS/TCF factors and are associated with increased TERT mRNA
(135). Therefore, point mutations in the TERT promoter could unravel a novel mechanism for
TERT reactivation in cancer cells. Recurrent mutations (Chr.5:1,295,228(C>T) or (CC>TT),
Chr.5:1,295,250 C>T, Chr.5:1,295,242_1,295,243CC>TT) have been identified in 19 % of
cancers (136).
Figure 15: Telomerase reverse transcriptase (TERT) promoter mutations identified in tumor samples and
potential E-twenty-six (ETS) binding sites (137)

Arrows indicate locations of mutations identified (red: potential de novo ETS binding site generated,
green: does not generate binding site.

1.3.2 Epigenetic induction

The TERT gene has a GC rich promoter which contains three CpG islands [53, 78]. Methylation
of gene promoters is generally known to repress transcription; however, several studies revealed
complex methylation patterns for the active/inactive TERT promoter. The TERT promoter region
has a CpG island (positions −1100 to +150), that is mostly hypermethylated through specific
DNA methyltransferases in cancer cells [79]. The TERT promoter between −150 and +150
represents a usual pattern of gene expression. Absence of methylation causes constitutive
expression particularly in this region, hypermethylation of 5′ TERT promoter prevents binding of
methylation sensitive CTCF repressor to the first exon; however, partial hypomethylation of core
promoter is required for TERT transcription. Thus, TERT promoter methylation represents a
unique model for transcription in which hypermethylation of cytosine islands causes inhibition of
TERT expression and this differs among different cell types [79, 80]. Hypermethylation of the
TERT core promoter results in low telomerase activity and better survival rate in B cell chronic
lymphocytic leukemia [81]. On the contrary, several studies reported that hypermethylation in
telomerase active cancer cells and hypomethylation of normal tissues may inhibit binding of
repressor elements to the TERT promoter region and enhance transcriptional activity.
The transcriptional regulation of the TERT gene occurs at multiple levels by various positive and
negative factors or signaling pathways. The TERT promoter interacts with different transcription
factors, responds to numerous physiological or oncogenic signals, and integrates these diverse
and dynamic inputs to set the TERT mRNA output (138).

1.4 TERT transcription regulation

There are various mechanisms regulating TERT expression at the transcriptional level. The most
famous is the binding of activating (e.g., c-MYC, SP1, STAT3, NF-κB, and ETS) or repressing
(e.g., MAD, p53, and WT1) transcription factors to the promotor region. These transcriptional
agents to TERT could be controlled by DNA methylation (CpG sites) in the TERT
Hypermethylation Oncological Region (THOR) (Figure 16).

Two main hotspot mutations within TERT promoter, -146C > T (C250T) and -124C > T (C228T)
upstream of the transcription start site generate new E-twenty-six (ETS) binding sites, leading to
GABP recruitment and, eventually, TERT transcription. Alternatively spliced variants of TERT,
which do not have telomerase activity, could be also generated. Most tissues and organs express
no or very low levels of TERT mRNA, dependent on histone markers that are correlated with
passive or active transcription in many cells. The transcription can also be regulated by different
miRNAs at the 3’UTR that inhibit translation of TERT (Figure 16) (133).

Figure 16: Mechanisms of TERT transcription regulation (133)

 1.5 TERT reactivation impact

Transcription from the TERT promoter is highly regulated and is the primary means of
controlling telomerase levels in diverse cell types and cellular states (139). Hayflick limit defines
the number of possible cell divisions and depends on the length of chromosomal telomeres,
which decreases in standard cells with every cell division. In the simulation, the default Hayflick
limit of a normal stem cell is 72 as an approximation of the realistic number between 50 and 70
(140). This ‘Hayflick limit’ (the finite proliferation potential of primary human cells) is
overcome by forced overexpression of TERT or through the introduction of transcription-
activating and cancer-associated mutations in the TERT promoter.

It has been shown that cultured human keratinocytes or mammary epithelial cells also lack TERT
and encounter telomere shortening-based roadblocks to proliferation. In contrast to the silenced
expression of TERT in these cultured human cell types, TERT is strongly upregulated in human
T cells during mitogenic stimulation. This upregulation is presumably important for long-term
clonal expansion of lymphocytes during immune responses. Consistent with this notion,
telomerase-knockout mouse lymphocytes with short telomeres show impaired proliferative
responses when exposed to mitogens (141)

1.6 TERT-based immunotherapy

The development of telomerase inhibitors has been challenging and thus far there are no
clinically approved strategies exploiting this cancer target (142). Therapies based on inhibiting
telomerase reverse transcriptase activity require an extended period of treatment before
anticancer effects are exerted due to their reliance on the gradual attrition of telomeres with each
cell division. This may make them unsuitable for use as first line therapy and increase the
potential for evolution and outgrowth of resistant clones (142).

The presence of TERT, as the rate-limiting step in the activation of telomerase, is a prerequisite
for carcinogenesis. Recent evidence has shown that the recently developed TERT-based therapies
are successful in cancer treatment. Moreover, telomere length can impact the efficacy of
telomerase-directed therapy. Time taken for telomerase inhibitors to exert anticancer effects is
expected to depend on initial telomere length since the length of the shortest telomere in a cell
dictates the onset of telomere dysfunction (143,144).
Studies regarding the regulation of TERT were initiated soon after research into the transcription,
translation, transport processing of TERT was begun. These methodologies have the common
goal of downregulating TERT expression at multiple stages of biogenesis. Examples of these
approaches include antisense oligonucleotides, dominant-negative TERT, and hammerhead-like
nucleases (144).

TERT promoter-driven suicide gene therapy is based on genes that encode proteins that control
the replication of microbial enzymes or oncolytic viruses that convert a prodrug into a toxic
substance. This therapy is also based on effective delivery systems, such as liposomes and
adenovirus vectors and aims to rapidly kill TERT-positive cells in tumors versus normal cells.
However, efficient delivery of gene therapy is a major challenge, and immunological reactions
may limit the dosing of the vector system. Furthermore, there is increasing evidence that the
TERT antigen recognized in both an MHC-I and -II restricted fashion elicits a CD4+ and CD8+
T lymphocyte response. In the immune response, dendritic cells are the most efficient antigen-
presenting cells. They have been used extensively to activate antitumor effector T and B
lymphocytes through pulsing with TERT RNA, DNA and an adenoviral vector containing the
TERT gene to specifically eradicate autologous TERT-positive tumor cells (Figure 17) (144).

Figure 17: Three TERT-based strategies for killing tumor cells (144)
2. micro-RNAs in Bladder cancer
2.1 microRNAs biogenesis and functions

microRNAs (miRNAs) are small noncoding RNAs that play important roles in post
transcriptional gene regulation. In animal cells, miRNAs regulate their targets by translational
inhibition and mRNA destabilization (145). Interestingly, numerous studies reaffirm the
existence of mature miRNA in the nucleus, and nucleus-cytoplasm transport mechanism has also
been illustrated and showed that maturation and the expression of some miRNA can be regulated
by other miRNAs in the nucleus (146,147). Moreover, rising evidence are showing that miRNAs
can also regulate gene expression at multiple levels including chromatin architecture,
transcription, RNA editing, RNA stability, and translation. miRNAs can activate or suppress
gene expression by targeting noncoding regulatory regions in gene promoter such as TATA box
motif and CpG island region, and epigenetic status such as DNA methylation (148).

MicroRNAs are crucial regulators of gene expression and promising candidates for biomarker
development. miRNA detection is of considerable significance in both disease diagnosis and in
the study of miRNA function. The importance of miRNA itself is due to the complicated
regulatory functions it plays in various life processes and its close relationship with some
diseases. Traditional methods for miRNA detection do not meet the current demands, so various
novel methods have been developed with a special focus on sensitivity and specificity (149).

miRNAs act as imperfect sequence guides to recruit a ribonucleoprotein (RNP) complex to the
complementary RNA. Using this paradigm, microRNAs act in a similar manner to other RNA
components of RNPs, namely to provide a sequence-specific binding component to allow the
RNP to act on a particular target. At the simplest level, microRNAs act to decrease the
expression of mRNAs that contain stretches of sequence complementary to the microRNA. This
function can be likened to the function of endogenous or synthetic short interfering RNA
(siRNA). However, microRNA function is more complicated and nuanced than this ‘on-off’
model would suggest (150,151).

Terms like silencing and even targeting suggest that repression of the target by a microRNA is
either ‘on’ or ‘off’. Generally, microRNAs do not act to completely silence their target genes, but
rather decrease expression. Similarly, microRNAs themselves are sometimes considered in this
binary model to either be on or off. Indeed, there are examples where microRNAs have
remarkable tissue specificity (e.g., miR-122 in the liver or miR-124 in the brain). There are also
instances of strong induction of microRNAs at specified developmental times (e.g., let-7 in
C.elegans development). However, more often microRNAs are expressed in multiple cell and
tissue types and have different expression levels, but rarely fit the dichotomous scheme of ‘on or
off’ (150).

miRNAs maintain cell homeostasis by negative gene regulation. Proper controlling of miRNA
expression is required for a balanced physiological environment, as these small molecules
influence almost every genetic pathway from cell cycle checkpoint, cell proliferation to
apoptosis, with a wide range of target genes. Deregulation in miRNAs expression correlates with
various cancers by acting as tumor suppressors and oncogenes. Although promising therapies
exist to control tumor development and progression, there is a lack of efficient diagnostic and
therapeutic approaches for delineating various types of cancer. The molecularly different tumors
can be differentiated by specific miRNA profiling as their phenotypic signatures, which can
hence be exploited to surmount the diagnostic and therapeutic challenges (152).

miRNAs affect all of six hallmarks of malignant cells: 1) self-sufficiency in growth signals (let-7
family), 2) insensitivity to anti-growth signals (miR-17-92 cluster), 3) evasion from apoptosis
(miR-34a), 4) limitless replicative potential (miR-372/373 cluster), 5) angiogenesis (miR-210),
and 6) invasion and metastases (miR-10b) (153). The pattern of miRNA expression can be
correlated with cancer type, stage, and other clinical variables, so that miRNA profiling can be
used as a tool for cancer diagnosis and prognosis (154). Several studies have provided
compelling evidence that alterations in transcriptional activators or repressors cause abnormal
pri-miRNA transcription in cancer. For instance, expression of the miR-34 family genes (miR-
34a, miR-34b, and miR-34c) are controlled by the transcription factor p53, reflecting the
importance of the p53 functional status in predicting miR-34 expression in human cancers. In
contrast, the miR-143/145 cluster is repressed by oncogenic RAS signaling that induces
tumorigenesis, while the c-Myc oncogenic transcription factor transactivates expression of the
miR-17~92 cluster (also known as oncomiR-1) and promotes cancer progression (155).
 2.2 miRNAs in bladder cancer

In bladder cancer, non-coding RNAs (ncRNAs), particularly, microRNAs , long non-coding

RNA (lncRNAs), and circular RNAs (circRNAs) were found to be associated with disease
occurrence and development (156). In 2011, Zhu et al. identified 226 differentially expressed
miRNAs in bladder cancer; of these, 182 were found to be upregulated while 44 were
downregulated (157). More recently, in a case-control study, Usuba et al., proposed a
combination of 7 miRNA (7‐miRNA panel: miR ‐6087, miR ‐6724 ‐5p, miR ‐3960, miR ‐1343 ‐5p,
miR‐1185‐1‐3p, miR‐6831‐5p and miR‐4695‐5p) that could discriminate bladder cancer from
non‐cancer and other types of tumors with the highest diagnostic accuracy (sensitivity: 95%;
specificity: 87%) regardless of the stage and grade of bladder cancer (158). In another study
aiming to identify a panel of miRNA signature that can predict aggressive bladder cancer
phenotype from normal nonaggressive counterpart using miRNA expression levels, Inamoto et
al., have shown that downregulation of six miRNAs were associated with high risk bladder
cancer (hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-4324, hsa-miR-34b-5p,
and hsa-miR-135a-3p) and upregulation of three were shown to be protective (hsa-miR-145-5p,
hsa-miR-29c-3p, and hsa-miR-33b-3p) (Table 4) (159).

Low miR-100 expression correlated with longer progression-free survival and cancer-specific
overall survival in NMIBC, and higher levels of miR-100 were associated with negative FGFR3
protein expression (160,161). Another miRNAs that plays an important role in bladder cancer
tumorigenesis is the tumor suppressor miR-138-5p. Among seven other miRNAs with the
potential to interact with TERT ( miR-29c-3p, miR-124-3p, miR-133a-3p, miR-133b, miR-150-
5p, miR-378a-3p and miR-422a) miR-138-5p was found to potentially inhibit TERT expression
and possibly targets PD-L (162,163). Additionally, low expression of miR-138 characterized
recurrent tumors and higher expression levels were associated with longer recurrence-free
survival in patients with NMIBC (160). Moreover, Many studies have linked miRNAs with PD1
and PD-L1 expressions (miR-138-5p, miR-34a-5p, miR-138-5p, the miR-200 family, miR-424
and miR-513) expressions and, indirectly, to resistances to immunotherapy by affecting PD-
1/PD-L1 interactions and T-cell functions modulation (Table 4) (164).
Table 4: Dysregulation of miRNA transcription in cancer (155).

1.1 miRNAs as diagnosis and prognosis biomarkers

1.1.1 miRNAs in diagnosis

Advances in miRNA analysis methods resulted in numerous recent publications on miRNAs as

putative biomarkers (165). miRNA-guided diagnostics is a powerful molecular approach for
evaluating clinical samples through miRNA detection and/or visualization. To date, this
approach has been successfully used to diagnose, manage, and/or monitor a wide range of
neoplastic and non-neoplastic diseases (166).

Numerous studies have shown altered miRNA profiles in multiple cancer types. Some miRNAs
have already been validated as diagnostic or prognostic biomarkers in various malignant
diseases, demonstrating their clinical utility and their potential in the field of personalized
medicine and diagnostics (167).

The most promising role of miRNAs is that of a potential biomarker. Evidence suggests that they
could play an essential role as biomarkers in cancer through exosome-mediated intercellular
communication, in neurology for the diagnosis of Alzheimer’s disease, for patients with spinal
cord injury, epilepsy or neurodegenerative ailments. It could also be used in other fields like
cardiology, as a faster and more accurate means of diagnosis for acute cardiovascular disease or
heart failure and in the case of infectious diseases for the diagnosis of sepsis (168).

In regard to the diagnosis role of miRNAs in BC, based on genome-wide serum miRNA analysis
by Miseq sequencing followed by evaluations in the training and validation sets with reverse
transcription quantitative real-time PCR assays from serum samples of 250 patients with bladder
cancer and 240 controls, a six-miRNA panel (miR-152, miR-148b-3p, miR-3187-3p, miR-15b-
5p, miR-27a-3p and miR-30a-5p) for the diagnosis of bladder cancer was developed. The
corresponding sensitivities of this panel for Ta, T1 and T2–T4 were 90.00, 84.85 and 89.36%,
significantly higher than those of urine cytology, which were 13.33, 30.30 and 44.68%,
respectively suggesting that a serum miRNA signature may have considerable clinical value in
diagnosing bladder cancer (169).
 1.1.2 miRNAs in prognosis

Unique miRNA expression profiles have been observed in various cancer types at different
stages, suggesting their potential as diagnostic and prognostic biomarkers. Due to their tumor-
specific and tissue-specific expression profiles, stability, robust clinical assays for detection in
serum as well as in formalin-fixed tissue samples, miRNAs have emerged as attractive
candidates for prognostic applications (170).

miRNAs can be used as a reliable biomarker for bladder cancer prognosis. In a large meta-
analysis performed on 26 studies that involved 2753 patients, results suggested that many
miRNAs expression aberration may predict prognosis in patients with BC. It has been shown that
microRNAs, particularly miR-21, miR-143, miR-155, miR-214, and miR-222, could serve as
meticulous follow-up markers for early detection of progression or recurrence and even useful
therapeutic targets for the treatment (171).

Yin et al. have recognized differentially expressed miRNAs between normal bladder tissue and
bladder cancer samples in the TCGA-BCLA dataset. They could construct a 21-miRNA
signature which was significantly correlated with the overall survival of bladder cancer patients.
Moreover, those miRNAs were associated with the prognosis of patients with bladder cancer,
and inspirational ideas for the future basic and clinical exploration (172).

1.2 Tissue variation expression of miRNAs

Hundreds of miRNAs are found to be aberrantly expressed in bladder cancer (173). Until the
present, miRNA changes in bladder tumor tissues have been the most widely reported among
three types of biological samples. Interestingly, apart from the miRNAs that share the same
variation trends in different types of samples, a large number of BC-related miRNAs show
disparate changes in different sample types. For example, miR-342-3p is found to be upregulated
in blood but downregulated in the patients’ urine. Similarly, miR-200c is widely reported to be
over-expressed in tumor tissues, while it is expressed to a lesser degree in the urine samples.
Since most research on bladder cancer is based on tissue samples, these differences suggest that
urine or circulating miRNAs may be disparately expressed, and they may work through totally
different mechanisms compared to tissue miRNAs (174). This disparity rises more question
about the miRNA and specimens sampling in cancer research (Figure 18).
 Figure 18: Ddiagram of multiple reported miRNAs in three biological samples in bladder cancer (174)

1.3 miRNAs-based therapeutics

MicroRNA are detectable in tissues and biofluids such as plasma, serum and urine. The easy and
non-invasive detection of miRNAs are making of them relevant clinical biomarkers (175). Plus,
miRNAs can represent an alternative or/and complementary therapeutic tool by direct
intravesical injections.

As FDA-approved small RNA drugs start to enter clinical medicine, ongoing studies for the
miRNA class of small RNAs expand its preclinical and clinical research applications (176).
miRNA therapeutics is a significant area for RNA therapeutics for biopharmaceuticals. It is
considered as “next-generation” therapeutics. The common miRNA market size was estimated at
$ 160.5 million in 2017 and is expected to grow drastically (177).
 In tumors, miRNAs are involved in a variety of chemoresistance-
related signaling pathways to regulate tumor resistance. Currently,
miRNA-based therapeutic strategies include the use of agomir-
miRNA, miR-101 mimic, and miRNA inhibitors. Extensive cell
experiments have demonstrated the potential of these strategies to
reverse chemoresistance in chemotherapy. miRNAs also have
potential value in the treatment of chemotherapy resistance in
bladder cancer (178).

siRNA or miRNA specific delivery to target cell populations using

approaches of nanobiotechnology is just beginning and looks
promising (179). Moreover, it has been shown that intravesical
administration of exogenous miR-145 is a valid therapy for bladder
cancer in a human bladder cancer xenograft model. (180) and thus a
promising therapeutic strategy that should be investigated in bladder
cancer patients.

Regulation of miRNA Expression by DNA Binding Factor

Mechanism/Function/
Factor miRNA Cancer Type
Clinical Correlation

miR-34a, miR-34b miR-34c Cell cycle arrest, apoptosis & senescence Various types of cancers
Transcriptional activation by p53
miR-145 Apoptosis Various cancers like prostate cancer
Transcriptional repression of miR- Various cancers like Pancreatic,
Transcriptional repression by RREB1 miR-143/145 cluster
143/145 cluster Colorectal Adenocarcinoma
Regulation by C/EBPβ, beta-
miR-145 Various cancers like Renal cancer
catenin/TCF4, FOXO1 &
FOXO3
Inverse correlation between MN1 and
Regulation by MN1 miR-20a, miR-181b acute myeloid leukemia (AML) patients
miRNAs
Controls the expression of E2F1, THBS1, Various types of cancer, including B-
miR-17~92 cluster
Cell CTGF, & PTEN lymphoma & Breast cancer
Transcriptional activation by Myc Nasopharyngeal carcinoma &
miR-200c, miR-26, miR-
Suppresses the expression of their genes
29, miR-30, let-7
Lymphoma

miR-210
HIF1α Repression of initiation of tumor growth Various cancers like Head & neck tumor
miR-155

ZEB1 & ZEB2 miR-200 family Various cancers

Suppression of miR-221/222
miR-221/222 Breast cancer
expression by NcoR/SMRT
Repression by ER
complex
miR-515 Increased levels of oncogenic SK1 Breast cancer
 miR-125b, miR-21, miR- Prostate cancer & Hematological
Oncogenic role
221/222, miR-27a, miR- malignancies
Androgen/AR
32

miR-135a, miR-141 Tumor suppressive role Prostate cancer

miR-141, miR-23, miR-320, let-
Progesterone receptor /PR Breast & ovarian cancer
7
Glucocorticoids/ GR miR-15, miR-16, miR-223 Incresead expression of miRNA Leukemia cell lines

Regulation of miRNA Expression by Epigenetic Alteration

Mechanism/Function/
Factor miRNA Cancer Type
Clinical Correlation

Promoter hypermethylation miR-127 Increassed expression of BCL6 Bladder Cancer

Breast, Colon, Liver, Leukemias &
Promoter hypermethylation miR-124-1 Activation of , CDK6
Lymphomas
Endometrial
Promoter hypermethylation miR-129-2 Upregulation of SOX4
Gastric cancer

miR-34a
Promoter hypermethylation Gastric, Prostate & Colon cancer
miR-34b/c
Bladder, breast, non-small lung cancer,
CpG methylation miR-200 Inactivation
leukemia