O iginal S udy
Combined Angiogenesis and P oli e a ion
Ma ke s’Exp essions as Long-Te m P ognos ic
Fac o s in Renal Cell Cance
Juha P. Vi man,
1,2
Pe i Bono,
3
Tiina H. Luukkaala,
4
Kaisa L. Sunela,
5
Paula M. Kujala,
6
Pi kko-Liisa I. Kellokumpu-Leh inen
1,5
Abs ac
The p ognos ic ole o MIB-1, BCL-2, VEGFR3, and CD31 exp ession was e ospec i ely e alua ed in 224 enal
cell cance (RCC) pa ien s. The combina ion o high MIB-1/low BCL-2 was wi h poo su i al compa ed wi h
low MIB-1/high BCL-2, and he combina ion o low VEGFR3/low CD31 was also associa ed wi h poo su i al
compa ed wi h high VEGFR3/high CD31. These molecula exp essions migh be aluable in planning he
ollow-up o RCC pa ien s.
Objec i es: The aim o his s udy was o e alua e he exp ession o MIB-1, BCL-2, VEGFR3, and CD31 and hei
associa ions wi h long- e m su i al in pa ien s wi h enal cell cance (RCC). Pa ien s and me hods: This s udy
consis ed o 224 RCC pa ien s who unde wen adical neph ec omy om 1985 o 1995. Follow-up con inued o up o
o e 20 yea s. MIB-1 and BCL-2 exp ession we e analyzed alone, and addi ionally, he exp ession o MIB-1, BCL-2,
VEGFR3, and CD31 we e combined in pai s using he ollowing g oups: low/low, low/high, high/low, and high/high.
Resul s: Low BCL-2 exp ession (haza d a io [HR], 2.16; 95% confidence in e al [CI], 1.42-3.31; P<.001 compa ed
wi h high BCL-2 in uni a ia e analysis) and high MIB-1 exp ession (HR, 2.05; 95% CI, 1.32-3.19; P¼.001 in mul i-
a ia e analysis) we e ound o associa e o poo e su i al in RCC. In mul i a ia e analysis, he combina ion o high
MIB-1/low BCL-2 was associa ed wi h poo su i al compa ed wi h low MIB-1/high BCL-2 (HR, 3.20; 95% CI,
1.66-6.17; P¼.001), and he combina ion o low VEGFR3/high CD31 was associa ed wi h poo su i al (HR, 2.48;
95% CI, 1.29-4.78; P¼.007) compa ed wi h high VEGFR3/high CD31. Conclusions: Compa ed wi h high BCL-2
exp ession in combina ion wi h low o high MIB-1, VEGFR3, o CD31 exp ession, low BCL-2 exp ession in combi-
na ion wi h low o high MIB-1, VEGFR3, o CD31 exp ession has poo e su i al in he long- e m ollow-up o pa ien s
wi h RCC. Analysis o MIB-1, BCL-2, VEGFR3, and CD31 exp ession migh be a use ul addi ional ma ke o ailo he
ollow-up o RCC pa ien s.
Clinical Geni ou ina y Cance , Vol. 14, No. 4, e283-9 ª2015 The Au ho s. Published by Else ie Inc. This is an open access
a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Keywo ds: BCL-2, CD31, MIB-1, RCC, VEGFR3
In oduc ion
The mos powe ul p ognos ic ac o s o pa ien s wi h localized
enal cell cance (RCC) emain umo s age, umo g ade, and
clinical a iables such as pe o mance s a us and p esence o absence
o symp oms, and se um ma ke s a e used o de e mine he p og-
nosis o pa ien s wi h me as a ic RCC (mRCC).
1-3
P e iously,
se e al possible immunochemical ma ke s ha e been s udied o
p edic he su i al o pa ien s wi h RCC, bu none has a ained
s a us as an independen p ognos ic ma ke .
4-10
Vascula endo helial g ow h ac o ecep o 3 (VEGFR3) is
impo an o lymphangiogenesis in no mal si ua ions, and i is also
1
Uni e si y o Tampe e, School o Medicine, Tampe e, Finland
2
Depa men o Anes hesia, Tampe e Uni e si y Hospi al, Tampe e, Finland
3
Cance Cen e , Helsinki Uni e si y Cen al Hospi al, Helsinki, Finland
4
Science Cen e , Pi kanmaa Hospi al Dis ic and School o Heal h Sciences,
Uni e si y o Tampe e, Tampe e, Finland
5
Depa men o Oncology, Tampe e Uni e si y Hospi al, Tampe e, Finland
6
Depa men o Pa hology, Tampe e Uni e si y Hospi al, Fimlab Labo a o ies,
Tampe e, Finland
Submi ed: Jul 10, 2015; Accep ed: Dec 16, 2015; Epub: Dec 22, 2015
Add ess o co espondence: Juha P. Vi man, MD, Tampe e Uni e si y Hospi al
Depa men o Anes hesia, PO Box 2000, FI-33521 Tampe e, Finland
E-mail con ac : Vi man.juha.[email p o ec ed].fi
1558-7673/ª2015 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
h p://dx.doi.o g/10.1016/j.clgc.2015.12.014 Clinical Geni ou ina y Cance Augus 2016
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e283
ac i a ed in cance and inflamma ion.
11
I main ains bo h angio-
genesis and he lympha ic sys em, and he e o e, i is conside ed an
in e es ing he apeu ic a ge .
12
Ma ked dec eased VEGFR3 plasma
le els ha e been shown in pa ien s wi h RCC a e ea men wi h
mul i- a ge ed y osine kinase inhibi o compa ed wi h s able o
p og essi e mRCC.
13
S udies o VEGFR3 exp ession and i s asso-
cia ion wi h umo s age, g ade, and su i al in pa ien s wi h RCC
ha e shown conflic ing esul s.
7,9,14,15
CD31 is a membe o an immunoglobulin supe amily o cell
adhesion molecules ha a e exp essed on he su aces o se e al
blood and endo helial cells.
16,17
Highe exp ession o CD31 has
been associa ed wi h a lowe umo g ade and be e su i al in
pa ien s wi h RCC,
5
and ou p e ious s udy also showed an asso-
cia ion be ween CD31 exp ession and su i al bu no umo g ade
o s age.
9
Mic o essel densi y has shown o co ela e nega i ely o
p ognosis in a leas RCC s udies.
18-20
In addi ion, CD31 exp es-
sion is ound o associa e wi h undi e en ia ed mic o essels.
21
MIB-1 is a well-known cell p oli e a ion ma ke , while BCL-2 is
a ma ke o cell dea h.
22,23
The exp ession o hese ma ke s has been
s udied in pa ien s wi h RCC. Highe MIB-1 exp ession was ound
o be independen ly associa ed wi h poo e su i al and wi h a
ecu ence in hese pa ien s.
4,24
Low BCL-2 exp ession was asso-
cia ed wi h a highe umo s age and a poo e p ognosis in pa ien s
wi h RCC.
6
On he con a y, 2 s udies did no find associa ion wi h
BCL-2 exp ession and a p ognosis in RCC.
8,25
Al hough he all ma ke s men ioned abo e ha e been s udied, none
has been iden ified as an independen p ognos ic ac o . The e o e, he
aim o his s udy was o explo e VEGFR3, CD31, MIB-1, and BCL-2
exp ession sepa a ely and in combina ion and o de e mine hei
associa ions wi h long- e m su i al in pa ien s wi h RCC.
Pa ien s and Me hods
Pa ien Popula ion
This s udy included pa ien s wi h RCC who unde wen
neph ec omy om 1985 o 1995. The su ge ies we e pe o med a
Tampe e Uni e si y Hospi al o a Tampe e Hospi al, Tampe e,
Finland. All o he umo samples we e eclassified and e-e alua ed
using he Heidelbe g classifica ion and Fuh man g ading sys em
26,27
by an expe ienced u opa hologis (P.K.). A o al o 202 (90.2%)
clea cell enal cell ca cinoma (ccRCC), 12 (5.4%) papilla y RCC
(pRCC), 5 (2.2%) ch omophobe RCC, 2 sa coma oid RCC
(0.9%), and 1 (0.4%) unclassified RCC we e included in his s udy.
The umo samples we e classified as Fuh man g ade 1 o 2
(22; 9.8%), g ade 3 (114; 50.9%), and g ade 4 (88; 39.3%).
Clinical s age was assessed acco ding o he TNM 2002 Classifi-
ca ion o Malignan Tumo s,
28
and 79 (35.3%), 43 (19.2%), 61
(27.2%), and 39 (17.4%) o pa ien s wi h RCC we e TNM s ages
1, 2, 3, and 4, espec i ely. The pa ien s’basic cha ac e is ics a e
desc ibed in a able in ou p e ious s udy.
9
Due o poo immunos aining, 13 (5.8%) pa ien s in he MIB-1
g oup, 3 (1.3%) in he BCL-2 g oup, 15 (6.7%) in he MIB-1/
BCL-2 g oup, 21 (9.4%) in he MIB-1/VEGFR3 g oup, 21
(9.4%) in he MIB-1/CD31 g oup, 15 (6.7%) in he BCL-2/CD31
g oup, and 14 (6.3%) in he VEGFR3/CD31 g oup we e excluded
om he analyses.
A e su ge y, he pa ien s’ ollow-up and ea men we e pe -
o med acco ding o s anda d clinical p ac ice a ha ime. The
median ollow-up ime was 5.4 yea s, wi h a ange o 0 o 21.7
yea s. Tampe e Uni e si y Hospi al and he Na ional Boa d o
Medicolegal A ai s app o ed he esea ch p o ocol and use o he
umo samples.
Immunos ainings
Sec ions we e depa a finized, and an igen e ie al was achie ed
by hea ing he sec ions in a mic owa e o en o 2 7 minu es in 10
mM is/10 mM e hylenediamine e aace ic acid (pH 9.0). Fo acid
Ki-67 an igen immunos aining, he monoclonal an ibody MIB-1
(IgG1, Immuno- ech S. A., Ma eille, F ance) was used a 1:110
dilu ion. Coun e s aining was accomplished using 0.4% e hyl g een
in ace a e bu e . The s aining o MIB-1 was e alua ed by isual
es ima ion and by using a compu e -assis ed image analysis sys em
(CAS-200 So wa e; Bec on Dickinson, Pa sippany, NJ, USA). The
MIB-1 index was defined as he pe cen o cells wi h immunopo-
si i i y in he nuclei. We fi s e alua ed pa ien s be ween 1990 and
1995 by isual es ima ion; only defini ely b own nuclei we e
eco ded as posi i e, and he same samples we e e alua ed using a
CAS-200. Spea man’s co ela ion be ween he isual es ima ion and
CAS-200 so wa e was excellen (0.826; P<.001). Samples om
pa ien s seen be ween 1985 and 1990 we e analyzed by CAS-200,
and he esul s o compu e -assis ed image analysis we e used o
s a is ical analysis.
Monoclonal mouse an ihuman BCL-2 oncop o ein clone 124
(Dako, Glos up, Denma k) was used a 1:60 dilu ion. Sec ions
we e sligh ly coun e s ained by hema oxylin, and s aining o
BCL-2 was analyzed semiquan i a i ely. S ainings we e quan i a ed
by in ensi y (0-3) and he pe cen a ea o exp ession (0%-100%)
and by mul iplying hese figu es o ob ain s aining sco es (0-300).
Immunohis ochemis y o CD31 (1:200, No ocas a Labo a-
o ies L d, Newcas le upon Tyne, UK) was pe o med on o malin-
fixed, pa a fin-embedded issue sec ions as a pa o a issue
mic oa ay. B iefly, he sec ions we e depa a finized wi h xylen and
ehyd a ed in g aded alcohol, ea ed in an au ocla e in 10 mmol/l
sodium ci a e (pH 5.0) o 2 minu es, and washed wi h phospha e
bu e ed saline. P ima y an ibody was incuba ed a 4C o e nigh ,
and an ibody binding was de ec ed by Vec as ain ABC ki eagen s
(Vec o Labo a o ies, Bu lingame, CA, USA). Diaminobenzidine
was used as he ch omogen. The slides we e coun e s ained wi h
hema oxylin and eosin, and moun ed.
VEGFR-3 was s ained wi h he 9D9 an ibody (a mouse mAb
agains he ex acellula domain o human VEGFR-3; a kind gi
om P o esso Ka i Ali alo, Helsinki, Finland) a he concen a ion
o 10
m
/L as desc ibed in de ail p e iously.
29
Mic o essel densi y was quan ified as he numbe o CD31-
posi i e o VEGFR-3-posi i e mic o essels pe high-powe field
a 250 (field o iew 0.407 mm
2
, including he whole issue
mic oa ay co e) using a Lei z Labo lux 12 b igh -field mic oscope
(Lei z We zla GmbH, Ge many). Two fields wi h he highes
densi y o essels we e coun ed, and an a e age o 2 sco es was
epo ed. Sco ing was pe o med in a blinded manne . Specimens
we e analyzed independen ly by wo in es iga o s.
S a is ical Me hods
S a is ical analyses we e pe o med using IBM SPSS S a is ic o
Windows e sion 21.0 (IBM Co p, Amonk, NY, USA; eleased
e284
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Clinical Geni ou ina y Cance Augus 2016
MIB-1, BCL-2, VEGFR3 and CD31 as P ognosis Fac o s in RCC
2010). The di e ences be ween ca ego ical a iables we e es ed
using he Pea son
c
2
es . The Cox p opo ional haza ds models
we e used in age- and gende -adjus ed uni a ia e and mul i a ia e
su i al analyses. In he mul i a ia e model, all dependen a iables
we e en e ed simul aneously in o he model. Fu he mo e, su i al
was analyzed using he Kaplan-Meie su i al es ima ion me hod.
All P- alues unde 0.05 we e conside ed s a is ically significan .
Resul s
Clinical Cha ac e is ics
Ou coho included 132 men (58.9%) and 92 woman (41.1%).
The pa ien s’median age a he ime o neph ec omy was 65 yea s
(in e qua ile ange, 55.9-71.9 yea s), and he median su i al ime
was 5.6 yea s (in e qua ile ange, 1.6-11.9 yea s).
MIB-1 Exp ession
Only one sample had nega i e s aining; he emaining samples
had posi i e exp ession o MIB-1. The median MIB-1 exp ession
alue was 1.36, and he highes exp ession alue was 40.9. The
MIB-1 exp ession alues we e di ided in o low (<1.36) and high
(1.36), acco ding o he median s aining esul . App oxima ely
hal o he ccRCC cases (96 samples; 50.3%) showed low exp es-
sion. Th ee (30%) papilla y RCCs (pRCC) had low and 7 (70%)
had high exp ession. Fou (80%) ch omophobe RCCs had low and
1 (20%) had high exp ession. The single collec ing duc umo and
he single unclassified RCC showed high exp ession. One sa co-
ma oid ype o RCC showed low exp ession, and he o he
exhibi ed high exp ession. The e was no associa ion be ween his-
ologic ype o RCC and MIB-1 exp ession (low o high; P¼.38).
Low exp ession o MIB-1 was obse ed in 10 (45.5%) g ade 1 o 2
umo s, and high exp ession was seen in 12 (54.5%); 55 (51.4%)
and 52 (48.6%) g ade 3 umo s and 39 (47.6%) and 43 (52.4%)
g ade 4 umo s showed low and high exp ession o MIB-1,
espec i ely. The e was no associa ion be ween low/high MIB-1
exp ession and umo g ade (P¼.81). Fo umo s ages 1, 2, 3,
and 4, he dis ibu ion o low/high MIB-1 exp ession was 43
(57.3%)/32 (42.7%), 23 (54.8%)/19 (45.2%), 23 (40.4%)/34
(59.6%), and 15 (41.7%)/21 (58.3%), espec i ely (P¼.19).
BCL-2 Exp ession
Se en y-fi e (33.9%) umo samples had nega i e s aining o
BCL-2. The median exp ession o BCL-2 was 30, and he highes
alue was 300. BCL-2 s aining was di ided in o low (0-30) and high
(31-300) exp ession g oups, based on he median alue. Immuno-
s aining o BCL-2 in ccRCC showed no di e ences; 102 (51.3%)
samples had low and 97 (48.7%) had high exp ession. Ten (83.3%)
pRCC had high and 2 (16.7%) had low BCL-2 exp ession. Th ee
(60%) ch omophobe RCCs had low and 2 (40%) had high
exp ession. Bo h sa coma oid RCCs had low immunos aining, as
did he single unclassified RCC, and he single collec ing duc RCC
had high immunos aining. Fishe exac es showed P¼.037 on
c oss- abula ion be ween low/high BCL-2 exp ession and di e en
his ological ypes o RCC. Fo umo g ades 1 o 2, 3, and 4, he
BCL-2 exp ession was low in 6 (28.6%), 49 (43.8%), and 56
(63.6%) cases and high in 15 (71.4%), 63 (56.3%), and 32
(36.4%) cases, espec i ely (P¼.002). Low exp ession was
obse ed in 30 (39.0%), 19 (43.2%), 37 (60.7%), and 25 (65.8%)
umo samples in umo s ages 1, 2, 3, and 4, espec i ely, and high
exp ession was obse ed in 47 (61.0%), 25 (58.8%), 24 (39.3%),
and 13 (34.2%) samples, espec i ely (P¼.011).
Exp essions o VEGFR3 and CD31
The exp ession o VEGFR3 and CD31 and hei dis ibu ions
wi h umo g ade and s age we e p esen ed in ou p e ious s udy.
9
B iefly, nei he VEGRF3 exp essions no CD31 exp essions showed
associa ion wi h umo s age o g ade.
Combina ions o MIB-1, BCL-2, VEGFR3, and CD31
Exp essions
The exp essions o MIB-1, BCL-2, VEGFR3, and CD31 we e
di ided in o wo g oups (low and high). MIB-1, BCL-2, and CD31
we e ca ego ized acco ding o hei median exp ession le els o 1.36,
30, and 18, espec i ely, as ollows: MIB-1 low (<1.35) and high
(1.35); BCL-2 low (0-30) and high (30-300); and CD31 low
(18) and high (>18). VEGFR3 exp ession was di ided in o low
(no posi i e essels) and high (posi i e essels). The c oss- abula ion
be ween MIB-1/BCL-2 exp ession and VEGFR3/CD31 exp ession
is desc ibed in Table 1.
Uni a ia e Analysis
Age- and gende -adjus ed uni a ia e analysis showed associa ions
be ween umo s age, g ade, and exp essions o MIB-1 and BCL-2
wi h su i al; o s age 4 compa ed wi h s age 1 disease: haza d a io
(HR), 13.8; 95% confidence in e al (CI), 7.18-26.7; P<.001; o
s age 3 compa ed wi h s age 1 disease: HR, 4.37; 95% CI, 2.29-
8.35; P<.01; o s age 2 compa ed wi h s age 1 disease: HR, 2.62;
95% CI, 1.27-5.41; P¼.007; o g ade 4 compa ed wi h g ades 1
o 2: HR, 9.31; 95% CI, 2.23-38.8; P¼.002; o g ade 3
compa ed wi h g ades 1 o 2: HR, 4.91; 95% CI, 1.18-20.4;
P<.001; o high MIB-1 compa ed wi h low MIB-1: HR, 1.76;
95% CI, 1.16-2.68; P¼.008; o low BCL-2 compa ed wi h high
BCL-2: HR, 2.16; 95% CI, 1.42-3.31; P<.001. Cox eg ession
uni a ia e analysis was also pe o med o pai s o ma ke s (MIB-1,
BCL-2, VEGFR3, and CD31) and all possible a ia ions o hei
exp essions (low/low, low/high, high/low and high/high); he esul s
a e summa ized in Table 2. E e y combina ion o MIB-1,
VEGFR3, o CD31 exp ession wi h high BCL-2 exp ession
showed s a is ically be e su i al compa ed wi h combina ions
wi h low BCL-2 exp ession. All combina ions o MIB-1/BCL-2
we e significan ly associa ed wi h su i al; o high MIB-1/high
Table 1 C oss-Tabula ion o MIB-1/BCL-2 Exp essions and
VEGFR3/CD31 Exp essions
MIB-1/
BCL-2
VEGFR3/CD31
To al
High/High low/low High/low low/High
n (%) n (%) n (%) n (%) n (%)
Low/high 26 (13) 10 (5) 12 (6) 9 (4.5) 57 (28.5)
High/high 18 (9.0) 6 (3.0) 15 (7.5) 4 (2.0) 43 (21.5)
Low/low 12 (6.0) 13 (6.5) 13 (6.5) 7 (3.5) 45 (22.5)
High/low 10 (5.0) 6 (3.0) 19 (9.5) 20 (10.0) 55 (27.5)
To al 66 (33.0) 35 (17.5) 59 (29.5) 40 (20) 200 (100)
The esul s a e indica ed in bo h numbe s and pe cen o cases, P¼.002 (Pea son
c
2
es )
Juha P. Vi man e al
Clinical Geni ou ina y Cance Augus 2016
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e285
BCL-2: HR, 2.13; 95% CI, 1.10-4.12; P¼.025; o low MIB-1/
low BCL-2: HR, 3.02; 95% CI, 1.62-5.62; P¼.001; o high
MIB-1/low BCL-2: HR, 3.51; 95% CI, 1.94-6.36; P<.001,
compa ed wi h low MIB-1/high BCL-2. The emaining combina-
ions showed no significan associa ion wi h su i al. Uni a ia e
analysis o VEGFR3 and CD31 exp ession wi h umo s age and
g ade showed ha low CD31 exp ession had poo e p ognosis in
RCC pa ien s bu VEGFR3 exp ession had no associa ion wi h
su i al in RCC. The esul s a e desc ibed ou p e ious s udy.
9
Mul i a ia e Analysis
Mul i a ia e Cox haza d eg ession analysis was pe o med
including umo g ade, umo s age, and exp essions o MIB-1
and BCL-2 simul aneously in o he model wi h age and gende ,
desc ibed in Table 3. S a is ically significan combina ions
MIB-1/BCL-2 and VEGFR3/CD31 in age- and gende -adjus ed
uni a ia e analyses we e es ed wi h umo s age and g ade in
mul i a ia e analysis. Highe umo s age showed poo e su i al,
and he combina ion o MIB-1/BCL-2 almos eached s a is ically
significan associa ion in su i al o all combina ions; he esul s
a e summa ized in Table 4.
Kaplan-Meie Analysis
Kaplan-Meie (KM) su i al analysis was used o illus a e MIB-1
and BCL-2 exp ession and he exp ession o he combina ions
desc ibed abo e. Inc eased BCL-2 exp ession and low MIB-1
exp ession showed be e su i al in KM cu es compa ed wi h
low BCL-2 and high MIB-1 exp essions (Figu es 1A and 1B).
Lowe BCL-2 exp ession in combina ion wi h low o high MIB-1,
VEGFR3, o CD31 exp ession showed poo e su i al compa ed
wi h any combina ions wi h high BCL-2 exp ession. The pla eau
phase, a ime a e diagnosis when pa ien s ha e no RCC- ela ed
mo ali y, was es ima ed by Kaplan-Meie su i al cu e analysis
and was ound o be 1.2 yea s o low g ade (1-2) umo s. Fo
highe umo g ades (3 and 4), he pla eau occu ed almos 10 yea s
la e , a 12.4 and 9.9 yea s, espec i ely. Fo s ages 1, 2, 3, and 4,
he pla eau phases we e eached wi hin 4, 8.5, 12.4, and 9.9 yea s,
espec i ely. No RCC- ela ed dea hs we e obse ed a e 9.9 and
12.4 yea s in umo s wi h low and high MIB-1 exp ession and a e
9.9 and 12.4 yea s in umo s wi h high and low BCL-2 exp ession,
espec i ely. O e hal o pa ien s ha ing exp ession combina ions
o low MIB-1/high BCL-2, low MIB-1/high VEGFR3, high BCL-2
and low/high VEGFR3, o high BCL-2 and low/high CD31 we e
ali e 10 yea s a e neph ec omy. KM cu es o he combina ions o
MIB-1/BCL-2, VEGFR/CD31, and BCL-2/VEGFR3 exp ession
a e shown in Figu es 2A-C.
Table 2 Age- and Gende -Adjus ed Uni a ia e Associa ions o
MIB-1, BCL-2, VEGFR3, and CD31 Exp essions
N
Age- and Gende -Adjus ed
Uni a ia e
HR [95% CI] P
MIB-1/BCL-2
Low/high 58 1.00
High/high 47 2.13 [1.10-4.12] .025
Low/low 45 3.02 [1.62-5.62] .001
High/low 59 3.51 [1.94-6.36] <.001
MIB-1/VEGFR3
Low/high 55 1.00
Low/low 48 1.65 [0.91-3.01] .10
High/low 46 1.87 [1.02-3.43] .042
High/high 54 2.36 [1.32-4.22] .004
MIB-1/CD31
Low/high 62 1.00
Low/low 41 1.45 [0.80-2.63] .22
High/low 59 1.83 [1.07-3.11] .026
High/high 41 2.14 [1.20-3.82] .01
BCL-2/VEGFR3
High/high 60 1.00
High/low 45 1.10 [0.57-2.13] .77
Low/low 52 2.37 [1.35-4.15] .003
Low/high 52 2.71 [1.55-4.76] <.001
BCL-2/CD31
High/high 62 1.00
High/low 44 0.91 [0.46-1.76] .78
Low/high 41 2.15 [1.21-3.83] .009
Low/low 62 2.38 [1.41-4.02] .001
VEGFR3/CD31
High/high 70 1.00
High/low 62 1.10 [0.65-1.88] .75
Low/high 35 2.02 [1.14-3.60] .017
Low/low 43 2.22 [1.29-3.82] .004
Cox eg ession models we e used, showing esul s by haza d a ios (HR) wi h 95% confidence
in e als (CI).
Table 3 Age- and Gende -Adjus ed Mul i a ia e Associa ions
o Tumo G ade, Tumo S age, and Exp essions o
MIB-1 and BCL-2
N
Age- and Gende -Adjus ed
Mul i a ia e
HR [95% CI] P
G ade
1-2 22 1.00
3 114 2.51 [0.59-10.69] .21
4 88 6.45 [1.50-27.8] .012
S age
1 79 1.00
2 44 2.96 [1.40-6.24] .004
3 61 3.58 [1.81-7.08] <.001
4 39 14.51 [7.13-29.55] <.001
MIB-1
Low 104 1.00
High 107 2.05 [1.32-3.19] .001
BCL-2
High 110 1.00
Low 111 1.40 [0.91-2.21] .12
Cox eg ession models we e used, showing esul s by haza d a ios (HR) wi h 95% confidence
in e als (CI).
MIB-1, BCL-2, VEGFR3 and CD31 as P ognosis Fac o s in RCC
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Clinical Geni ou ina y Cance Augus 2016
Discussion
In his s udy, we explo ed he exp ession o MIB-1, BCL-2,
VEGFR3, and CD31 and hei associa ions wi h su i al in pa ien s
wi h RCC. Ou s udy consis ed o 224 pa ien s, o whom all umo
samples we e e-e alua ed and eclassified by a qualified u opa-
hologis (P.K.). All da a we e collec ed di ec ly om he o iginal
medical eco ds. The su i al da a we e ob ained om he Finnish
Cance Regis y; hus we e e y eliable.
Clinicians oday ha e a be e unde s anding o molecula
pa hway abno mali ies, his ological sub ypes, and new mo pholog-
ical a ian s o RCC.
30
The imp o ed knowledge migh aid in
he disco e y o new ea men s o pa ien s wi h RCC.
31,32
The
s anda d ollow-up p ac ice o pa ien s wi h RCC is no clea and
has been deba ed in he li e a u e, and he endency is owa d mo e
indi idual ea men and managemen o RCC. We u gen ly need
new molecula ma ke s o plan hese pa ien s’indi idual ea men s
and ollow-up.
In he p esen s udy, he exp ession o he p oli e a ion ma ke s
MIB-1 and BCL-2 alone and wi h he angiogenesis ma ke s
VEGFR3 and CD31, and hei associa ions wi h su i al, was
explo ed in pa ien s wi h RCC. We classified he exp ession o
MIB-1, BCL-2, VEGFR3, and CD31 in o low and high exp ession
g oups acco ding o hei immunos aining sco es. High MIB-1
exp ession was associa ed wi h poo e disease- ee su i al and
o e all su i al in pa ien s wi h RCC in a p e ious s udy.
33
Simila ly, ou s udy showed ha low MIB-1 exp ession was inde-
penden ly associa ed wi h a be e p ognosis in hese pa ien s.
Highe BCL-2 exp ession in combina ion wi h any low o high
exp ession o MIB-1, VEGFR3, o CD31 showed be e su i al
compa ed wi h combina ions wi h low BCL-2 exp ession. P e ious
s udies ha e also ound ha inc eased BCL-2 exp ession was
associa ed wi h a be e p ognosis o pa ien s wi h RCC.
6
Ou
uni a ia e analysis showed ha a combina ion o he p oli e a ion
ma ke MIB-1 and an an i-apop osis ma ke BCL-2 had a s a is i-
cally significan associa ion wi h su i al in pa ien s wi h RCC.
An i-apop o ic p o eins such as BCL-2 a e o en o e exp essed in
cance , while apop o ic p o eins a e de egula ed.
34
RCC is, in
Table 4 Age- and Gende -Adjus ed Mul i a ia e Associa ions
o MIB-1/BCL-2 and VEGFR3/CD31 Exp essions Wi h
S age and G ade
n
Age- and Gende -Adjus ed
Mul i a ia e
HR [95% CI] P
MIB-1/BCL-2
Low/high 57 1.00
Low/low 45 1.60 [0.83-3.08] .16
High/high 42 2.14 [1.07-4.29] .032
High/low 55 3.20 [1.66-6.17] .001
VEGFR3/CD31
High/high 66 1.00
Low/low 58 0.92 [0.52-1.63] .79
High/low 40 1.28 [0.70-2.43] .46
Low/high 35 2.48 [1.29-4.78] .007
S age
1 67 1.00
2 40 2.60 [1.28-5.25] .008
3 57 2.58 [1.40-4.73] .002
4 35 8.85 [4.47-17.5] <.001
G ade
1-2 20 1.00
3 102 1.44 [0.48-4.24] .51
4 78 3.25 [1.09-9.73] .035
Cox eg ession models we e used, showing esul s by haza d a ios (HR) wi h 95% confidence
in e als (CI).
Figu e 1 (A) Kaplan-Meie Cu es o MIB-1 Exp ession (Low/High). (B) Kaplan-Meie Cu es o BCL-2 Exp ession (Low/High)
Juha P. Vi man e al
Clinical Geni ou ina y Cance Augus 2016
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e287

gene al, a highly ascula umo , and angiogenesis is e y impo an
o umo g ow h and sp ead. His ological umo nec osis is shown
o be associa ed wi h poo e su i al in RCC.
35
O e exp ession o
BCL-2 was shown o p e en nec osis,
36
which migh be one o he
explana o y ac o s o he imp o ed su i al o pa ien s wi h RCC
and highe BCL-2 exp ession. MIB-1 exp ession was also signifi-
can ly associa ed wi h su i al in mul i a ia e analysis, in which he
classical p ognos ic ac o s o umo s age and g ade we e also
included. Ou p e ious s udy showed ha highe CD31 exp ession
was associa ed wi h a be e p ognosis in pa ien s wi h RCC
compa ed wi h low CD31 exp ession.
9
This p esen s udy showed
ha highe CD31 exp ession had no s ong associa ion wi h su -
i al, whe eas high BCL-2 had a s ong associa ion wi h be e
su i al wi h all combina ions o MIB-1, VEGFR3, and CD31
exp essions.
VEGFR3 is impo an o bo h angiogenesis and lympha ic
main enance,
12
whe eas CD31 is in ol ed in issue egene a ion,
and i s exp ession has been shown in ascula umo s.
16
This s udy
showed ha he combina ion o high VEGFR3/high CD31 was
associa ed wi h a be e p ognosis compa ed wi h low VEGFR3/low
CD31. This may indica e ha pa ien s wi h be e lymphangio-
genesis and ac i e issue egene a ion ha e a be e p ognosis.
We also examined associa ions be ween combina ions o MIB-1
and BCL-2 exp ession, which a e ela ed o cell g ow h and
apop osis, and combina ions o VEGFR3 and CD31 exp ession,
which a e ela ed o umo angiogenesis. C oss- abula ion showed a
significan associa ion be ween hem, as shown in Table 1. Dis i-
bu ions we e in he same line as su i al analysis showed; howe e ,
4.5% o cases wi h low MIB-1/high BCL-2, which was associa ed
wi h be e su i al, had low VEGFR3/high CD31, which was
associa ed wi h poo e su i al; in addi ion, 5% o cases wi h high
MIB-1/low BCL-2, which was associa ed wi h a poo e p ognosis,
had high VEGFR3/high CD31, which was associa ed wi h a be e
p ognosis. Based on hese esul s, we canno clea ly indica e clea
associa ions be ween he exp essions o hese combina ions o
ac o s; pe haps he e a e o he ac o s o which we a e no ye awa e
ha a ec umo g ow h, dea h, and umo angiogenesis.
This p esen s udy included 224 consecu i e pa ien s whose
RCC was diagnosed om 1985 o 1995. Du ing ha ime, adio-
logical examina ions we e pe o med less equen ly han hey a e
cu en ly. O e 50% o RCCs a e cu en ly de ec ed inciden ally.
30
This migh be he eason ha ou s udy popula ion consis ed o
only 22 (9.8%) pa ien s wi h g ade 1 o 2 umo and 79 (35.3%)
pa ien s wi h s age 1 umo . Ou s udy showed an associa ion
be ween highe Bcl-2 exp ession and pRCC, bu no associa ion
be ween MIB-1 exp ession and his ological ype o RCC. This
migh be explained by he ac ha he majo i y his ological ype in
ou s udy was ccRCC (90.2%) and his ep esen s a highe p o-
po ion han is gene ally ound in RCC.
26,37,38
Due o inadequa e
immunos aining, we excluded 3 o 21 pa ien s (1.3%-9.4%) om
he analyses. The excluded po ion was, howe e , mino and
he e o e had an insignifican e ec on he esul s.
Ou s udy showed ha o e 50% o pa ien s wi h low MIB-1 o
high Bcl-2 exp ession we e ali e a he end o long- e m ollow-up.
The su i al pla eau was eached al eady a e 1.2 yea s in pa ien s
wi h g ade 1 o 2 umo s, whe eas o o he g ades and s ages, he
MIB-1 and Bcl-2 exp ession g oups’pla eaus we e achie ed be ween
8.5 and 12.4 yea s. Addi ionally, 50% o pa ien s wi h low MIB/
high BCL-2, low MIB-1/high VEGFR3, high BCL-2 and low/high
VEGFR3, o high BCL-2 and low/high CD31exp ession su i ed
10 yea s a e he diagnosis o RCC. These da a showed ha all
pa ien s wi h RCC who we e ali e 10 yea s a e neph ec omy had
an excellen p ognosis. The ollow-up guidelines o RCC ha e been
ecen ly discussed in he li e a u e.
32
Smi h e al. showed ha nea ly
one- hi d o RCC ecu ences we e missed when pa ien s we e
ollowed acco ding o he Ame ican U ological Associa ion o
Na ional Comp ehensi e Cance Ne wo k guidelines.
39
New
molecula ma ke s o hei combina ions migh be needed o
imp o e he assessmen o ecu ence isk and o ailo ea men
and ollow-up o pa ien s wi h RCC. MIB-1, BCL-2, VEGFR3,
Figu e 2 (A) Kaplan-Meie Cu es o Combina ions o MIB-1 and BCL-2 Exp essions (Low/High). (B) Kaplan-Meie Cu es o
Combina ions o BCL-2 and CD31 Exp essions (Low/High). (C) Kaplan-Meie Cu es o Combina ions o BCL-2 and VEGFR3
Exp essions (Low/High)
MIB-1, BCL-2, VEGFR3 and CD31 as P ognosis Fac o s in RCC
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Clinical Geni ou ina y Cance Augus 2016
and CD31 migh be use ul o indi idual ollow-up o pa ien s wi h
RCC and should be es ed in p ospec i e ials.
Conclusion
These da a showed ha low BCL-2 alone o in any combina ion
wi h low o high MIB-1, VEGFR3, o CD31 exp ession was
associa ed wi h poo e su i al in pa ien s wi h RCC. Low MIB-1
exp ession was an independen p edic o o be e p ognosis in
pa ien s wi h RCC. Low VEGFR3/low CD31 exp ession was
associa ed wi h poo su i al compa ed wi h high VEGFR3/high
CD31 exp ession. These ma ke s migh be use ul o planning he
ollow-up o pa ien s wi h RCC.
Clinical P ac ice Poin s
The e a e no molecula ma ke s o p edic su i al in pa ien s
wi h RCC. We e alua ed he exp essions o MIB-1, BCL-2,
VEGFR3, and CD31 and hei ole in he p ognosis o RCC.
This s udy showed ha low MIB-1 and high BCL-2 exp essions
we e associa ed wi h imp o ed su i al in RCC.
High VEGFR3/high CD31 exp ession showed be e su i al
compa ed wi h low VEGFR3/high CD31 exp ession.
Molecula ma ke s migh be use ul o planning pa ien s’
ollow-up.
Disclosu e
The au ho s ha e s a ed ha hey ha e no conflic s o in e es .
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