FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients
Aim: The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder cancer patients and to assess the relationship between their mutational status and clinicopathological variables. Materials and Methods: The FGFR3 and TP53 mutations were de...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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irk-123456789-1453822019-01-22T01:23:31Z FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients Smal, M.P. Rolevich, A.I. Polyakov, S.L. Krasny, S.A. Goncharova, R.I. Original contributions Aim: The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder cancer patients and to assess the relationship between their mutational status and clinicopathological variables. Materials and Methods: The FGFR3 and TP53 mutations were detected by the SNaPshot method and PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. Results: The activating FGFR3 mutations were found in 71 (47.3%) whereas TP53 mutations were observed in 31 (20.7%) urothelial carcinomas. FGFR3-mutant tumors significantly correlated with lower tumor stage and grade, papillary form of bladder cancer and the absence of metastases while TP53-mutant tumors were strongly associated with higher tumor stage and grade as well as the presence of metastasis. We also found significant inverse correlation between FGFR3 mutations and TP53 alterations in urothelial carcinomas (p=0.03). Four possible genotypes were observed in the whole studied cohort, namely FGFR3mut/TP53wt (41.3%), FGFR3wt/TP53wt (38%), FGFR3wt/TP53mut (14.7%), and FGFR3mut/TP53mut (6%). Tumors with FGFR3wt/TP53wt genotype comprised the subgroup, in which all stages and grades were equally distributed. Conclusions: Our findings confirm the alternative role of FGFR3 and TP53 mutations in the development of bladder cancer. Together these two genetic markers are attributed to 62% of the tumors studied. Tumors with both wild type genes included urothelial carcinomas of all stages and grades and may develop through another genetic pathway. To elucidate complete molecular profile of bladder tumors further additional studies are needed. Key Words: bladder cancer, FGFR3 mutation, TP53 mutation, tumor genotype. 2014 Article FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients / M.P. Smal, A.I. Rolevich, S.L. Polyakov, S.A. Krasny, R.I. Goncharova // Experimental Oncology. — 2014. — Т. 36, № 4. — С. 246-251. — Бібліогр.: 25 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145382 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Smal, M.P. Rolevich, A.I. Polyakov, S.L. Krasny, S.A. Goncharova, R.I. FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients Experimental Oncology |
| description |
Aim: The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder cancer patients and to assess the relationship between their mutational status and clinicopathological variables. Materials and Methods: The FGFR3 and TP53 mutations were detected by the SNaPshot method and PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. Results: The activating FGFR3 mutations were found in 71 (47.3%) whereas TP53 mutations were observed in 31 (20.7%) urothelial carcinomas. FGFR3-mutant tumors significantly correlated with lower tumor stage and grade, papillary form of bladder cancer and the absence of metastases while TP53-mutant tumors were strongly associated with higher tumor stage and grade as well as the presence of metastasis. We also found significant inverse correlation between FGFR3 mutations and TP53 alterations in urothelial carcinomas (p=0.03). Four possible genotypes were observed in the whole studied cohort, namely FGFR3mut/TP53wt (41.3%), FGFR3wt/TP53wt (38%), FGFR3wt/TP53mut (14.7%), and FGFR3mut/TP53mut (6%). Tumors with FGFR3wt/TP53wt genotype comprised the subgroup, in which all stages and grades were equally distributed. Conclusions: Our findings confirm the alternative role of FGFR3 and TP53 mutations in the development of bladder cancer. Together these two genetic markers are attributed to 62% of the tumors studied. Tumors with both wild type genes included urothelial carcinomas of all stages and grades and may develop through another genetic pathway. To elucidate complete molecular profile of bladder tumors further additional studies are needed. Key Words: bladder cancer, FGFR3 mutation, TP53 mutation, tumor genotype. |
| format |
Article |
| author |
Smal, M.P. Rolevich, A.I. Polyakov, S.L. Krasny, S.A. Goncharova, R.I. |
| author_facet |
Smal, M.P. Rolevich, A.I. Polyakov, S.L. Krasny, S.A. Goncharova, R.I. |
| author_sort |
Smal, M.P. |
| title |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients |
| title_short |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients |
| title_full |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients |
| title_fullStr |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients |
| title_full_unstemmed |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients |
| title_sort |
fgfr3 and tp53 mutations in a prospective cohort of belarusian bladder cancer patients |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/145382 |
| citation_txt |
FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients / M.P. Smal, A.I. Rolevich, S.L. Polyakov, S.A. Krasny, R.I. Goncharova // Experimental Oncology. — 2014. — Т. 36, № 4. — С. 246-251. — Бібліогр.: 25 назв. — англ. |
| series |
Experimental Oncology |
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| first_indexed |
2025-07-10T21:32:54Z |
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2025-07-10T21:32:54Z |
| _version_ |
1837297237701427200 |
| fulltext |
246 Experimental Oncology 36, 246–251, 2014 (December)
FGFR3 AND TP53 MUTATIONS IN A PROSPECTIVE COHORT
OF BELARUSIAN BLADDER CANCER PATIENTS
M.P. Smal1, A.I. Rolevich2, S.L. Polyakov2, S.A. Krasny2, R.I. Goncharova1, *
1Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk 20072, Republic of Belarus
2N.N. Alexandrov National Cancer Center of Belarus, Department of Urology, Minsk 223040,
Republic of Belarus
Aim: The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder
cancer patients and to assess the relationship between their mutational status and clinicopathological variables. Materials and
Methods: The FGFR3 and TP53 mutations were detected by the SNaPshot method and PCR-single-strand conformational poly-
morphism analysis followed by DNA sequencing. Results: The activating FGFR3 mutations were found in 71 (47.3%) whereas
TP53 mutations were observed in 31 (20.7%) urothelial carcinomas. FGFR3-mutant tumors significantly correlated with lower
tumor stage and grade, papillary form of bladder cancer and the absence of metastases while TP53-mutant tumors were strongly
associated with higher tumor stage and grade as well as the presence of metastasis. We also found significant inverse correlation
between FGFR3 mutations and TP53 alterations in urothelial carcinomas (p=0.03). Four possible genotypes were observed
in the whole studied cohort, namely FGFR3mut/TP53wt (41.3%), FGFR3wt/TP53wt (38%), FGFR3wt/TP53mut (14.7%), and
FGFR3mut/TP53mut (6%). Tumors with FGFR3wt/TP53wt genotype comprised the subgroup, in which all stages and grades were
equally distributed. Conclusions: Our findings confirm the alternative role of FGFR3 and TP53 mutations in the development
of bladder cancer. Together these two genetic markers are attributed to 62% of the tumors studied. Tumors with both wild type
genes included urothelial carcinomas of all stages and grades and may develop through another genetic pathway. To elucidate
complete molecular profile of bladder tumors further additional studies are needed.
Key Words: bladder cancer, FGFR3 mutation, TP53 mutation, tumor genotype.
Bladder carcinoma is the most common mali-
gnancy of urinary tract. In Europe, as well as in Be-
larus, it is the 7th most frequent cancer in men [1, 2].
Urothelial cell carcinoma (UCC) comprises 90% of all
bladder cancer cases. Bladder tumors are morpho-
logically heterogeneous and can be categorized into
two groups: non-muscle invasive and muscle invasive.
At the time of diagnosis more than 75% of bladder
tumors are non-muscle invasive Ta, CIS, and T1 neo-
plasms [1]. The remaining percentage of patients (i.e.
20–25%) is diagnosed with muscle invasive bladder
cancer (MIBC) ≥Т2 who undergo cystectomy and have
a poor prognosis [3]. After transurethral resection,
most patients with non-muscle invasive bladder cancer
(NMIBC) (70–80%) develop recurrences within 5 years,
and some of them (10–20%) show progression into
MIBC which is considered potentially lethal. It provides
grounds to assume that NMIBC is a heterogeneous
group of cancers with different prognosis. A number
of researchers proposed the hypothesis that clinical and
morphological heterogeneity of bladder tumors is de-
termined by different molecular pathways of urothelial
carcinoma pathoge nesis [4–7]. Non-muscle invasive
bladder tumors are mainly characterized by high fre-
quency of activating FGFR3 missense mutations, op-
posed to high frequency of inactivating TP53 alterations
in MIBC. The prevalence of FGFR3 mutations in non-
muscle invasive low grade urothelial carcinomas and
their correlation with favourable prognosis suggested
FGFR3 mutation to be a potential molecular marker for
prognosis of NMIBC [8, 9]. Further molecular studies
of bladder cancer not only proved the existence of two
alternative genetic pathways of urothelial carcinoma
pathoge nesis but also revealed a more complex pic-
ture of bladder tumor mutability and genome instabi-
lity [10–12].
In our previous study of a prospective cohort of Be-
larusian patients with NMIBC and MIBC, we found a high
frequency (45.5%) of activating FGFR3 gene muta-
tions that was significantly associated with low stage
and low grade tumors [13]. To understand completely
the genetic basis of urothelial carcinoma, there is a need
to study the mutational status of other genes involved
in bladder cancer development. The aim of the present
study was to determine the frequencies of FGFR3 and
TP53 mutations in a prospective cohort of bladder
cancer patients and to find the relationship between
clinicopathological parameters of the disease and tu-
mor genotypes according to mutations in both genes.
MATERIALS AND METHODS
Рatients and tissue samples. We analyzed the tu-
mor tissues of 150 patients (115 men and 35 women)
with histologically confirmed UCC who were treated
at the Department of Urology of N.N. Alexandrov
National Cancer Center of Belarus between 2010 and
2013. The median age of the patients was 67.5 years.
Written informed consent was obtained from each pa-
tient. Fresh tissue samples and matched formalin-fixed
paraffin-embedded (FFPE) tissue samples were used
as biological material for analysis. Tumors were graded
Submitted: September 09, 2014.
*Correspondence: E-mail: R.Goncharova@igc.bas-net.by
Abbreviations used: MIBC — muscle invasive bladder cancer;
NMIBC — non-muscle invasive bladder cancer; PCR — polymerase
chain reaction; SSCP — single-strand conformation polymorphism;
UCC — urothelial cell carcinoma; wt/mut — wild type/mutant.
Exp Oncol 2014
36, 4, 246–251
Experimental Oncology 36, 246–251, 2014 (December) 247
according to the 1973 and 2004 WHO classification
and staged according to the tumor-node-metastasis
classification guidelines. Sociodemographic and clini-
cal information is presented in Table 1.
Table 1. Characteristics of the patients and tumor samples
Parameter Number of patients Frequency, %
Gender
male 115 76.7
female 35 23.3
Age, years
< 50 13 8.7
51–60 28 18.7
61–70 44 29.3
≥ 71 65 43.3
Mean age (m ± SD) 66.1 ± 10.2
Stage
Та 16 10.7
Т1 81 54.0
Т2 25 16.6
Т3 13 8.7
Т4 15 10.0
Grade (WHO 1973)
G1 54 36.0
G2 65 43.3
G3 31 20.7
Grade (WHO 2004)
low 84 56.0
high 66 44.0
Multiplicity
solitary 52 34.7
multiple 97 64.7
missing 1 0.6
Shape
papillary 110 73.3
non-papillary 40 26.7
Tumor size, cm
≤ 3 82 54.7
> 3 67 44.7
missing 1 0.6
Smoking
yes 56 37.3
no 87 58.0
missing 7 4.7
DNA extraction. Genomic DNA was isolated from
both fresh tissue samples and deparaffinized FFPE
tissue sections (5–10 μm) by using a standard phenol-
chlorophorm method.
FGFR3 mutation analysis. FGFR3 mutational
status was analyzed using a previously described
highly sensitive SNaPshot assay [14] that allows
screening for 11 point mutations (R248C, S249C,
G372C, S373C, Y375C, G382R, A393E, K652E, K652M,
K652Q, and K652T). Briefly, three regions comp rising
all known FGFR3 mutations were amplified in one
multiplex polymerase chain reaction (PCR), followed
by extension of internal primers for each mutation with
a labeled dideoxynucleotide. Extended primers were
separated by capillary electrophoresis in an automatic
sequencer ABI Prism 3500 (Applied Biosystems), and
the pre sence or absence of a mutation was defined
by the incorporated dideoxynucleotide.
TP53 mutation analysis. The regions comprising
exons 5–8 of the TP53 gene were amplified by PCR
using the primers described by Thongsuksai et al. [15].
PCRs were performed in a total volume of 15 μl con-
taining 100 ng genomic DNA, 1× buffer (100 mM Tris-
HCl, pH 8.3, 500 mM KCl), 0.2 μM of each primer,
2 mM MgCl2, 0.2 mM dNTPs, and 0.5 U Dream Taq DNA
Polymerase (Thermo Scientific). The cycling condi-
tions were as follows: denaturation at 94 °C for 5 min,
followed by 35 cycles of denaturation at 94 °C for 30 s,
annealing at 56 °C for 30 s, extension at 72 °C for 50 s,
with a final extension at 72 °C for 10 min on C1000 ther-
mal cycler (Bio-Rad).
For single-strand conformation polymorphism
(SSCP) analysis, 7 μl of PCR product were added
to 10 μl of a solution containing 95% formamide,
10 mM NaOH, 20 mM EDTA, 0.05% of xylene cya-
nol, and 0.05% of bromophenol blue. The mixture
was denatured at 100 °C for 7 min then immediately
cooled on ice and loaded onto a 10% acrylamide gel
(20 x 20 cm plate) containing 5% glycerol. Electropho-
resis was performed at 280 V constant power and run
for 7 h at 8 °C. After electrophoresis the gel was stained
with ethidium bromide.
Samples that showed band shift on SSCP gel were
subjected to direct sequencing, which was performed
using the Big Dye Terminator v 3.1 cycle sequencing
kit (Applied Biosystems) in accordance with the manu-
facturer’s instructions. All mutations were confirmed
by sequencing both DNA strands.
Statistical analysis. To assess the association
between FGFR3 and TP53 genetic alterations and clini-
copathological parameters of tumors Chi-square test,
Fisher’s exact test, or Mann — Whitney U test were
used when appropriate, and values of p < 0.05 were
considered statistically significant.
RESULTS
Frequency and spectrum of FGFR3 gene muta-
tions. Somatic point FGFR3 mutations were detected
in 71 (47.3%) of 150 UCCs, double mutations being
found in 6 tumors. Seven different missense muta-
tions, namely S249C, R248C, Y375C, G372C, A393E,
S373C, and К652М were identified. Among 6 cases
with double mutation, three tumors harbored S249C/
R248C alterations, while the other three had A393E/
R248C, S249C/Y375C and К652М/A393E combina-
tions. The frequency and distribution of all identified
FGFR3 mutations are shown in Table 2. The most
common changes were S249C (68.8%) and Y375C
(16.9%). The overall frequency of the mutations in co-
dons 248, 249, and 375 — the major hot spots in blad-
der tumors — was 93.5% of total mutations in FGFR3.
Table 2. Frequencies and spectrum of FGFR3 gene mutations
Exon Nucleotide
change
Amino acid
change
Number
of mutations, n
Frequency
of mutations, %
7 742С > Т R248C 6 7.8
7 746С > G S249C 53 68.8
10 1114G > Т G372C 1 1.3
10 1117A > Т S373C 1 1.3
10 1124A > G Y375C 13 16.9
10 1178C > A A393E 2 2.6
15 1955A > Т К652М 1 1.3
Frequency and spectrum of TP53 gene muta-
tions. TP53 alterations were found in 31 (20.7%)
tumors, out of which 37.8% were detected in exon 8,
followed by 35.2% in exon 7, 21.6% in exon 5 and 5.4%
in exon 6. Twenty five patients had only one mutation,
double TP53 mutation was observed in 6 (4%) cases.
Distribution of the TP53 alterations over 27 distinct
codons is shown in Table 3. All molecular changes
248 Experimental Oncology 36, 246–251, 2014 (December)
of the TP53 gene were presented by missense point
mutations except one patient, who had a silent mutation.
48.6% of the gene alterations were transitions and 51.4%
were transversions. No mutation leading to a premature
termination of the protein was found. The codons 248,
175, and 285 were mutational hot spots in bladder tumors.
Table 3. ТР53 gene mutations detected in bladder tumors
Sample Stage Grade Exon Codon Mutation Amino acid change
28 1 1 low 7 245 GGC-GTC Gly-Val
39 1 1 low 8 273 CGT-TGT Arg-Cys
73 1 1 low 7 246 ATG-ATA Met-Ile
8 280 AGA-ACA Arg-Thr
105 1 1 low 7 241 TCC-TGC Ser-Cys
101 1 2 low 5 175 CGC-CAC Arg-His
64 1 2 high 6 195 ATC-ACC Ile-Thr
1 1 3 high 5 175 CGC-CAC Arg-His
128 1 3 high 8 274 GTT-TTT Val-Phe
155 2 2 low 8 283 CGC-TGC Arg-Cys
22 2 2 high 6 196 CGA-CCA Arg-Pro
81 2 2 high 7 248 CGG-CAG Arg-Gln
121 2 2 high 8 271 GAG-CAG Glu-Gln
149 2 2 high 7 244 GGC-GAC Gly-Asp
15 2 3 high 8 285 GAG-AAG Glu-Lys
92 2 3 high 7 248 CGG-CAG Arg-Gln
130 2 3 high 5 139 AAG-GAG Lys-Glu
20 3 2 high 7 248 CGG-CGC Arg-Arg
8 280 AGA-ACA Arg-Thr
70 3 2 high 8 271 GAG-CAG Glu-Gln
8 291 AAG-AAC Lys-Asn
123 3 2 high 8 278 CCT-CTT Pro-Leu
110 3 3 high 8 285 GAG-AAG Glu-Lys
133 3 3 high 7 257 CTG-CCG Leu-Pro
156 3 3 high 5 159 GCC-GTC Ala-Val
5 179 CAT-AAT His-Asn
9 4 2 high 5 175 CGC-CAC Arg-His
49 4 2 high 7 248 CGG-CAG Arg-Gln
138 4 2 high 5 146 TGG-TGT Trp-Cys
8 4 3 high 7 245 GGC-TGC Gly-Cys
7 246 ATG-ATC Met-Ile
13 4 3 high 7 249 AGG-AGT Arg-Ser
124 4 3 high 5 132 AAG-AAC Lys-Asn
134 4 3 high 7 238 TGT-TTT Cys-Phe
140 4 3 high 8 267 CGG-CAG Arg-Gln
150 4 3 high 8 281 GAC-CAC Asp-His
8 285 GAG-CAG Glu-Gln
Association of FGFR3 and TP53 mutations with
clinicopathological parameters of tumors. Sociode-
mographical variables and morphologic features of tu-
mors according to FGFR3 and TP53 mutational status
are shown in Table 4. No correlation was found between
FGFR3 changes and various characteristics like age, gen-
der, smoking, multifocality and tumor size. At the same
time, statistically significant association of FGFR3 muta-
tions with papillary form of bladder cancer (р < 0.001) and
non-metastatic disease (р = 0.02) was revealed.
The frequency of FGFR3 mutations in non-muscle
invasive tumors was 57.7% against only 28.3% in mus-
cle invasive T2-T4 UCCs (p < 0.001). We found strong
correlation between FGFR3 mutations and tumor stage
(р < 0.001). FGFR3 changes had the highest frequency
(62.5%) in Tа tumors and the lowest (13.3%) in T4 car-
cinomas. Mutational status of the FGFR3 gene was also
significantly associated with tumor grade (р < 0.001).
The distribution of the frequency of FGFR3 alterations
according to tumor grade was as follows: 68.5% in G1,
47.7% in G2 and 9.7% in G3 tumors. Thus, non-muscle
invasive papillary low grade UCCs are characterized
by high frequency of FGFR3 alterations.
As to TP53 gene, no statistically significant diffe-
rence was noted in the distribution of TP53 mutations
on the basis of gender, age and smoking. However,
mutations were strongly related to non-papillary form
of bladder cancer, large tumor size, solitarity and
the presence of metastasis (Table 4). The frequency
of molecular changes of this gene was substantially
higher in MIBC (43.4%) than NMIBC (8.2%). A strong
association was found between TP53 mutations and
high tumor stage (р < 0.001). No TP53 mutation was de-
tected in Tа tumors, the frequency of the gene changes
increased from about 10% in T1 to 60% in T4 tumors.
Mutational status of the TP53 gene was significantly
related to tumor grade: a higher rate of TP53 altera-
tions was observed in the group of G3 (45.2%) tumors
compared to G2 (20%) and G1 (7.4%) tumors. Hence,
high frequency of TP53 genetic changes characterizes
high grade muscle invasive tumors of large size.
Table 4. Clinicopathological variables according to TP53 and FGFR3 mu-
tational status
Parameter
FGFR3 mutation
р
TP53 mutation
рyes,
n (%)
no,
n
yes,
n (%)
no,
n
Gender 0.87 0.55
male 54 (47.0) 61 22 (19.1) 93
female 17 (48.6) 18 9 (25.7) 26
Age, years 0.86 0.59
< 50 6 (46.2) 7 5 (38.5) 8
51–60 12 (42.9) 16 6 (21.4) 22
61–70 19 (43.2) 25 7 (15.9) 37
≥ 71 34 (52.3) 31 13 (20.0) 52
Stage <0.001 <0.001
Та 10 (62.5) 6 0 (0) 16
Т1 46 (56.8) 35 8 (9.9) 73
Т2 12 (48.0) 13 8 (32.0) 17
Т3 1 (7.7) 12 6 (46.2) 7
Т4 2 (13.3) 13 9 (60.0) 6
Grade (WHO 1973) <0.001 <0.001
G1 37 (68.5) 17 4 (7.4) 50
G2 31 (47.7) 34 13 (20.0) 52
G3 3 (9.7) 28 14 (45.2) 17
Grade (WHO 2004) <0.001 <0.001
low 53 (63.1) 31 6 (7.1) 78
high 18 (27.3) 48 25 (37.9) 41
Multiplicity 0.4 0.028
solitary 22 (42.3) 30 16 (30.8) 36
multiple 48 (49.5) 49 15 (15.5) 82
Shape <0.001 <0.001
papillary 64 (58.2) 46 13 (11.8) 97
non-papillary 7 (17.5) 33 18 (45.0) 22
Tumor size, cm 0.33 0.001
≤ 3 42 (51.2) 40 8 (9.8) 74
> 3 29 (43.3) 38 22 (32.8) 45
Metastasis 0.02 <0.001
yes 2 (15.4) 11 9 (69.2) 4
no 69 (50.4) 68 22 (16.1) 115
Smoking 0.66 0.41
> 20 years smoker 37 (50.0) 37 17 (23.0) 57
non-smoker/
< 20 years smoker 32 (46.4) 37 12 (17.4) 57
The strong associations between FGFR3 and
TP53 mutational status and tumor stage and grade
are clearly depicted in Fig. 1. An inverse relation was
found between FGFR3 mutations and TP53 altera-
tions (p = 0.037). Stage and grade information taken
together showed a high proportion of FGFR3 muta-
tions and TP53 alterations in tumors with low and high
malignant potential, respectively.
Analysis of tumor genotypes revealed that geno-
type FGFR3mut/TP53wt was the most prevalent, ac-
counting for 41.3% of UCCs; tumors FGFR3wt/TP53wt
lacking alteration in both genes were observed in 38%
of cases, and frequencies of genotypes FGFR3wt/
Experimental Oncology 36, 246–251, 2014 (December) 249
TP53mut and FGFR3mut/TP53mut were 14.7% and
6%, respectively. Interestingly, FGFR3 and ТP53 alte-
rations were almost mutually exclusive, and they coin-
cided in only 9 tumors of 150 (6%).
0
10
20
30
40
50
60
70
80
Та Т1 Т2 T3 T4
Fr
eq
ue
nc
y
(%
)
FGFR3mut
TP53mut
0
10
20
30
40
50
60
70
80
G1 G2 G3
Fr
eq
ue
nc
y
(%
)
a
b
Fig. 1 FGFR3 and TP53 mutations according to tumor stage (a)
and grade (b)
Tumor genotypes at various stages and grades are
shown in Fig. 2.
a
b
0
10
20
30
40
50
60
70
Та Т1 Т2-4
Fr
eq
ue
nc
y
(%
)
FGFR3mut/TP53wt
FGFR3wt/TP53mut
FGFR3mut/TP53mut
FGFR3wt/TP53wt
0
10
20
30
40
50
60
70
G1 G2 G3
Fr
eq
ue
nc
y
(%
)
Fig. 2 Tumor genotypes according to mutations in both
FGFR3 and TP53 at various stages (a) and grades (b)
In Tа tumors, more commonly observed genotypes
were FGFR3mut/TP53wt (62.5%) and FGFR3wt/
TP53wt (37.5%). In the group of T1 carcinomas,
the frequencies of various genotypes were as under:
FGFR3mut/TP53wt — 50.6%, FGFR3wt/TP53wt —
39.5%, FGFR3mut/TP53mut — 6.2% and FGFR3wt/
TP53mut — 3.7%. In invasive T2–4 tumors, genotypes
FGFR3wt/TP53mut (35.8%) and FGFR3wt/TP53wt
(35.8%) were the most prevalent, followed by geno-
types FGFR3mut/TP53wt (20.8%) and FGFR3mut/
TP53mut (7.5%).
Distribution of four genotypes according to tumor
grade showed that the majority of low grade UCCs had
FGFR3mut/TP53wt and FGFR3wt/TP53wt genotypes,
while in high grade carcinomas, genotypes FGFR3wt/
TP53wt and FGFR3wt/TP53mut were more common.
On the whole the data indicate that there is a strong
association of FGFR3 mutations with favorable dise-
ase parameters (low grade, non-muscle invasive),
and TP53 alterations with unfavorable tumor features
(high grade, muscle invasive). Patients carrying either
FGFR3 or TP53 mutation accounted for 62% of the whole
cohort studied. The group of UCCs that were wild type
for both genes represented a considerable part (more
than 35%) of tumors of all stages and grades.
DISCUSSION
To our knowledge, the first two pathway model
of urothelial carcinoma pathogenesis was proposed
by Spruck et al. [4]. Stratification of tumors into
groups was based on the differences in the frequen-
cies of TP53 mutations and chromosome 9 deletions.
Later, this model was modified by van Rhijn et al. [6]
by adding FGFR3 marker. The authors found an in-
verse relationship between FGFR3 and ТР53 mutabi-
lity and clinical morphological parameters of tumors
and/or disease-specific survival (a higher frequency
of FGFR3 mutations is indicative of high disease-
specific survival while elevated rate of TP53 alterations
indicates low disease-specific survival). This finding
suggested that mutations of corresponding genes
may serve as the molecular markers of good and poor
prognosis in bladder cancer patients.
We also have demonstrated an inverse relation
between FGFR3 and TP53 mutations in Belarusian
bladder cancer patients. In non-muscle invasive
papillary low grade tumors, we found high percentage
of FGFR3 mutations which is in accordance with previ-
ous studies in European [16, 17] and Belarusian popu-
lations [13]. In the present investigation, like in other
reports [18, 19], high frequency of TP53 alterations
was observed in advanced tumors.
Moreover, we analyzed the frequencies of geno-
types according to FGFR3 and TP53 mutational status
in the groups of tumors of different stages and grades.
In Ta group, no alteration of the TP53 gene was found.
However, a number of studies reported that TP53 mu-
tations may occur at this tumor stage as well, though
quite rarely [19, 20, 21].
In non-muscle invasive T1 tumors, we found the high
frequency of FGFR3mut/TP53wt genotype accounting
for more than 50%, while UCCs with FGFR3wt/TP53mut
250 Experimental Oncology 36, 246–251, 2014 (December)
genotype were observed in 3.7%. Conversely, the pre-
vious studies reported that <27% and >20% of T1 tu-
mors had FGFR3mut/TP53wt and FGFR3wt/TP53mut
genotypes, respectively [5, 19]. Such differences
in the frequencies of T1 tumor genotypes in Belarus
compared to Western Europe may be explained by high
percentage of muscle invasive disease at first diagnosis
in Belarus and further by prevalence of T1 UCCs among
the majority of patients with NMIBC. T1 group com-
prises tumors with different genotypes suggesting their
genetic heterogeneity which is reflected in the clinical
picture of the disease.
The genetic nature of tumors which are wild type
for both FGFR3 and TP53 genes (38% of UCCs in our
study) remains unclear. The tumors of this subgroup
can develop either through the third pathway of bladder
cancer pathogenesis, supposed by van Rhijn et al. [6],
or due to hyperexpression of the normal FGFR3 gene
observed by Tomlinson et al. in 42% of UCCs with wild
type FGFR3 [22]. Recent investigations on bladder
cancer revealed that its formation and development
may not be referred to just two main pathways based
on FGFR3 and TP53 alterations, respectively [23].
Whole-genome studies of different cancers found
a considerable number of somatic mutations in the tu-
mor genome [12] and it raised a problem of defining
the key drivers. The key mutations rather than accom-
panying (passenger) ones could be used in bladder
cancer diagnosis and prognosis. Detailed molecular
genetic analyses conducted by Lindgren et al. and Guo
et al. [10, 11] validated the role of FGFR3 and TP53 mu-
tations in UCC pathogenesis. Besides, these analyses
along with other data established the pathogenetic
role of mutations in several other genes, particularly
RB1, PIK3CA, KRAS, HRAS, NRAS, CDKN2A, and
TSC1. However, the distribution of RAS [10, 24, 25],
CDKN2A and TSC1 [10] mutations is independent
of tumor stage and grade, and thus, they are not of high
prognostic value.
According to whole-genome analysis, UCC was
divided into two molecular subtypes (MS1 and MS2),
differentiated by the mutational status and patterns
of gene expression [10]. MS1 tumors are characte-
rized by high frequency of activating FGFR3 mutations
which often coincide with PIK3CA alterations, whereas
TP53 and RB1 changes are more common in MS2 tu-
mors. When comparing mutational profiles of MS1 and
MS2 carcinomas with clinicopathological parameters,
the authors distinguished the subgroups of UCCs ac-
cording to the level of genome instability. Tumors with
FGFR3 mutations were found genetically more stable
than those with TP53 alterations, characterized by high
proportion of chromosome aberrations [10, 17].
Whole-genome and whole-exome sequen cing
of 99 bladder tumors [11] showed alterations in 37 genes
including well known FGFR3, TP53, RB1, PIK3CA,
KRAS, HRAS, and TSC1. Moreover, the authors identi-
fied the essential role of mutations in the genes involved
in sister chromatid cohesion and segregation process.
The frequency of genetic alterations affecting this pro-
cess was 32% and related to more aggressive tumor
phenotype. Besides, the high frequency of mutations
in the genes involved in cell cycle control, DNA repair
and chromatin post-translational modifications was
found. The role of these genes in determination of mo-
lecular subtypes of bladder tumors will be elucidated
by studying the association of mutations with genome
instability and clinicopathological parameters.
The data mentioned indicate a complex genetic
nature of UCCs. Our findings confirm the alternative
role of FGFR3 and TP53 mutations in non-muscle and
muscle invasive bladder tumors as well as their genetic
heterogeneity. Our data support the idea that tumors
with FGFR3wt/TP53wt genotype may represent the third
pathway of bladder cancer pathogenesis. Further study
of the molecular profile of UCC is needed to define mo-
lecular subtypes of bladder tumors, which are important
to improve bladder cancer diagnosis and prognosis.
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