EURURO Vol. 72 No. 3

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an advanced stage and grade [30_TD$DIFF] and in more recent
studies, MYC amplification was observed in about 13% of
MIBCs [5_TD$DIFF]
Inactivating mutations in genes encoding DNA repair
proteins were also relatively common in MIBCs [31_TD$DIFF] . The
most prevalent were inactivating mutations in ERCC2 (12%
of tumors) [5_TD$DIFF] , which were linked to sensitivity to
neoadjuvant cisplatin-based combination chemotherapy
[32_TD$DIFF] . Inactivating mutations in several other DNA repair
proteins were also linked to cisplatin sensitivity [33_TD$DIFF] .
Inactivating mutations in STAG2, a component of the
cohesin complex that functions in chromosome segrega-
tion, were common in NMIBCs and MIBCs [34_TD$DIFF] [35_TD$DIFF] , but
their biological significance remains unclear; the canonical
role of the cohesin complex would suggest that inactivation
of STAG2 should produce genomic instability, but the
significant enrichment of STAG2 mutations in low-grade
tumors that largely lack aneuploidy [35_TD$DIFF] argues against this
being their most relevant effect in bladder cancers [34_TD$DIFF]
[35_TD$DIFF] . Alternative mechanisms include alterations in high-
order chromatin organization and gene expression.
Activating mutations in the telomerase ( TERT1) promot-
er were common in both NMIBCs and MIBCs [36_TD$DIFF] [37_TD$DIFF] ,
making them attractive biomarkers for early detection of
recurrence [38_TD$DIFF] and potentially as therapeutic targets
across the course of disease progression.
Bladder cancers often contained DNA alterations involv-
ing oncogenes or tumor suppressor genes that regulate
activation of the Ras–MEK–ERK and PI3 kinase–AKT–mTOR
pathways. These pathways control progression through the
RB1-dependent G1-S cell cycle restriction point, anabolic
metabolism, and cell survival. Activating mutations in
FGFR3 were detected in up to 80% of NMIBCs and
approximately 15–20% of MIBCs, consistent with earlier
studies [39_TD$DIFF] . Preclinical studies demonstrated that
these FGFR3 mutations, which cause constitutive receptor
activation, functioned to promote proliferation via down-
stream activation of the ERKs [40_TD$DIFF] . Papillary and
nonpapillary cancers contained similar frequencies of
activating RAS mutations (5–10%) [41_TD$DIFF] which also function
to promote downstream ERK activation. RAS and FGFR3
mutations occurred in a mutually exclusive fashion [5_TD$DIFF] so
together they probably accounted for enhanced ERK
activation in almost 90% of NMIBCs. Although activating
FGFR3 mutations were less common in MIBCs, some MIBCs
contained activating FGFR3 fusions [42_TD$DIFF] . They also con-
tained activating mutations, fusions, or amplification of
genes encoding members of the epidermal growth factor
receptor (EGFR) family [15_TD$DIFF] , including the EGFR itself (about
10% of tumors), ERBB2 (about 10% of tumors), ERBB3 (about
10% tumors), and ERBB4 (about 6% of tumors). Other tumors
contained inactivating mutations in the RAS inhibitor, NF1
(over 10%) [28_TD$DIFF] ; so, together, these alterations probably
promoted RAS pathway activation in over 50% of MIBCs.
With respect to the PI3 kinase/AKT pathway, activating
PIK3CA mutations—predominantly in the region coding for
the helical domain, probably caused by APOBEC3B-mediat-
ed mutagenesis [20_TD$DIFF] —were common in both NMIBCs and
MIBCs. Amplification of AKT3 or inactivating mutations in
various negative regulators of the PI3 kinase/AKT/mTOR
pathway (including the lipid phosphatase PTEN) were also
observed, leading to predicted pathway activation in almost
75% of MIBCs [28_TD$DIFF] .
Mutations and/or amplification of transcription factors
implicated in urothelial terminal differentiation were found
in MIBCs [5_TD$DIFF] Amplifications of peroxisome proliferator
activator receptor-gamma (PPARG), GATA3, and SOX4 were
frequently detected, occurring in about 10–15% of tumors
[5_TD$DIFF] . Mutations in ELF3, RXRA, and KLF5 were also relatively
common, occurring in 5–10% of tumors [5_TD$DIFF] . Mutations in
FOXA1 were observed in about 5% of tumors, and deletion of
FOXQ1 occurred in about 10% of tumors [5_TD$DIFF] . Inactivating
mutations in NOTCH1 and NOTCH2 have also been reported
in MIBCs [43_TD$DIFF] , and preclinical studies in mouse models
suggested that they promoted tumor progression by
facilitating epithelial-to-mesenchymal transition (EMT)
[44_TD$DIFF] . Finally, mutations in the ubiquitin ligase and NOTCH
pathway regulator, FBXW7, occurred in about 7% of MIBCs
[5_TD$DIFF] . Although inactivating mutations in other develop-
mental pathways were less common, preclinical studies
have suggested that activation of the Wnt/b-catenin
pathway and downregulation of the sonic hedgehog
pathway also contribute to bladder cancer progression
[45_TD$DIFF] .
3.4. Molecular subtypes of bladder cancer
The identification and validation of molecular subtypes in
other malignancies provided the impetus to use transcrip-
tome profiling to search for molecular subtypes in bladder
cancers. The initial results established that unsupervised
analyses of gene expression could distinguish most NMIBCs
from most MIBCs [46_TD$DIFF] [47_TD$DIFF] Furthermore, early studies of
NMIBCs identified gene expression signatures associated
with disease aggressiveness using unsupervised analyses
[48_TD$DIFF] [49_TD$DIFF] . These studies showed the first indications of the
presence of major molecular subtypes in bladder cancer
[50_TD$DIFF] . One of our groups (M.H., Lund University, Lund,
Sweden) extended these findings by implicating differences
in[51_TD$DIFF] TP53 mutation frequencies and/or genomic instability to
the formation of these two major gene expression subtypes
[52_TD$DIFF] . Subsequently, they used a large cohort of NMIBCs and
MIBCs (n = 308) to identify additional subtypes within the
two major clusters [53_TD$DIFF] . The Lund classification revealed
that bladder cancers could be segregated into at least five
molecular subtypes, termed urobasal A (uroA), urobasal B
(uroB), genomically unstable (GU), infiltrated, and SCC like
(SCCL) [53_TD$DIFF] . The uroA and uroB tumors were characterized
by stratified expression of differentiation-associated bio-
markers reminiscent of what is observed in the normal
urothelium, whereas differentiation-associated biomarkers
displayed abnormal expression in the GU and SCCL tumors.
The SCCL subtype was characterized by expression of
squamous keratins (KRT5, KRT6, and KRT14) and keratini-
zation-associated genes [54_TD$DIFF] , and the SCCL and uroB tumors
were both enriched with various degrees of squamous
differentiation markers [55_TD$DIFF] . As its name implies, the
infiltrated subtype was characterized by the expression of
E U R O P E A N U R O L O G Y 7 2 ( 2 0 1 7 ) 3 5 4 – 3 6 5 357