PRECLINICAL STUDY
Biological sub yping o ea ly b eas cance : a s udy compa ing
RT-qPCR wi h immunohis ochemis y
Ralph M. Wi z
1
•Ha i Sih o
2
•Jo ma Isola
3
•Pa
¨i i Heikkila
¨
4
•
Pi kko-Liisa Kellokumpu-Leh inen
5
•Pa
¨i i Au inen
6
•Taina Tu peenniemi-Hujanen
7
•
Si kku Jy kkio
¨
8
•So i is Lakis
1
•Ko nelia Schlombs
9
•Ma k Laible
9
•
S e an Webe
10
•Sebas ian Eid
11
•Ugu Sahin
9
•Heikki Joensuu
12
Recei ed: 8 Ap il 2016 / Accep ed: 13 May 2016 / Published online: 24 May 2016
ÓThe Au ho (s) 2016. This a icle is published wi h open access a Sp inge link.com
Abs ac The biological sub ype o b eas cance in lu-
ences he selec ion o sys emic he apy. Dis inc ion
be ween luminal A and B cance s depends on consis en
assessmen o Ki-67, bu subs an ial in a-obse e and
in e -obse e a iabili y exis s when immunohis ochem-
is y (IHC) is used. We compa ed RT-qPCR wi h IHC in
he assessmen o Ki-67 and o he s anda d ac o s used in
b eas cance sub yping. RNA was ex ac ed om a chi al
b eas umou issue o 769 women andomly assigned o
he FinHe ial. Cance ESR1,PGR,ERBB2 and MKI67
mRNA con en was quan i a ed wi h an RT-qPCR assay.
Local pa hologis s assessed ER, PgR and Ki-67 exp ession
using IHC. HER2 ampli ica ion was iden i ied wi h
ch omogenic in si u hyb idiza ion (CISH) cen ally. The
esul s we e co ela ed wi h dis an disease- ee su i al
(DDFS) and o e all su i al (OS). qPCR-based and IHC-
based assessmen s o ER and PgR showed good conco -
dance. Bo h low umou MKI67 mRNA (RT-qPCR) and
Ki-67 p o ein (IHC) le els we e p ognos ic o a ou able
DDFS [haza d a io (HR) 0.42, 95 % CI 0.25–0.71,
P=0.001; and HR 0.56, 0.37–0.84, P=0.005, espec-
i ely] and OS. In mul i a iable analyses, cance MKI67
mRNA con en had independen in luence on DDFS (ad-
jus ed HR 0.51, 95 % CI 0.29–0.89, P=0.019) while Ki-
67 p o ein exp ession had no any in luence (P=0.266)
whe eas bo h assessmen s in luenced independen ly OS.
Luminal B pa ien s ea ed wi h doce axel-FEC had mo e
a ou able DDFS and OS han hose ea ed wi h ino el-
bine-FEC when he sub ype was de ined by RT-qPCR ( o
Elec onic supplemen a y ma e ial The online e sion o his
a icle (doi:10.1007/s10549-016-3835-7) con ains supplemen a y
ma e ial, which is a ailable o au ho ized use s.
&Ralph M. Wi z
[email p o ec ed]
Ha i Sih o
[email p o ec ed]
Jo ma Isola
[email p o ec ed]
Pa
¨i i Heikkila
¨
[email p o ec ed]
Pi kko-Liisa Kellokumpu-Leh inen
[email p o ec ed]
Pa
¨i i Au inen
[email p o ec ed]
Taina Tu peenniemi-Hujanen
[email p o ec ed]
Si kku Jy kkio
¨
[email p o ec ed]
So i is Lakis
[email p o ec ed]
Ko nelia Schlombs
[email p o ec ed]
Ma k Laible
[email p o ec ed]
S e an Webe
[email p o ec ed]
Sebas ian Eid
[email p o ec ed]
Ugu Sahin
[email p o ec ed]
Heikki Joensuu
[email p o ec ed]
1
STRATIFYER Molecula Pa hology GmbH, We hmanns .
1c, 50935 Cologne, Ge many
2
Labo a o y o Molecula Oncology, T ansla ional Cance
Biology P og am, Uni e si y o Helsinki, Helsinki, Finland
123
B eas Cance Res T ea (2016) 157:437–446
DOI 10.1007/s10549-016-3835-7
DDFS, HR 0.52, 95 % CI 0.29–0.94, P=0.031), bu no
when de ined using IHC. B eas cance sub ypes app oxi-
ma ed wi h RT-qPCR and IHC show good conco dance,
bu cance MKI67 mRNA con en co ela ed sligh ly be e
wi h DDFS han Ki-67 exp ession. The indings based on
MKI67 mRNA con en sugges ha pa ien s wi h luminal B
cance bene i mo e om doce axel-FEC han om
ino elbine-FEC.
Keywo ds B eas cance Molecula sub ypes Ki-67 
P edic ion Immunohis ochemis y RT-qPCR
Abb e ia ions
ESR1/ER Oes ogen Recep o alpha
CISH Ch omogenic in si u hyb idiza ion
Cq Quan i ica ion cycle
DDFS Dis an disease- ee su i al
FEC Fluo ou acil & epi ubicin,
cyclophosphamide chemo he apy
FFPE Fo malin ixed pa a in embedded
GOI Gene o in e es
HR Haza d a io
IHC Immunohis ochemis y
mRNA Messenge ibonucleic acid
MKI67/
Ki-67
Ma ke o p oli e a ion Ki-67
OS O e all su i al
PGR/PgR P oges e one ecep o
REF Re e ence gene
RT-qPCR Re e se ansc ip ion quan i a i e eal- ime
polyme ase chain eac ion
ERBB2/
HER2
Human epide mal g ow h ac o ecep o 2
In oduc ion
Biological sub yping o b eas cance is an in eg al pa o
he s anda d e alua ion o pa ien s diagnosed wi h b eas
cance . Sub yping can be done wi h gene exp ession a ays
[1], bu he molecula sub ypes a e equen ly app oxi-
ma ed wi h immunohis ochemis y (IHC) due o i s wide
a ailabili y and low cos . Howe e , assays o cance
oes ogen ecep o (ER), p oges e one ecep o (PgR) and
human epide mal g ow h ac o ecep o -2 (HER2)
exp ession by IHC ha e an up o 20 % isk o disco dan
o e oneous esul s [2,3], and making a dis inc ion
be ween luminal A and luminal B b eas cance equi es
assessmen wi h he p oli e a ion ma ke Ki-67, which is
p one o high in a- and in e -obse e assessmen a i-
abili y [4,5].
In his s udy, we compa ed assessmen o b eas cance
key bioma ke s, ER, PgR, HER2 and Ki-67 quan i a i ely
using RT-qPCR wi h hei assessmen using IHC o in si u
hyb idiza ion as a pa o he clinical ou ine in b eas
cance sub yping and p edic ion o pa ien ou come. We
hypo hesized ha quan i ying Ki-67 wi h RT-qPCR migh
esul in mo e obus ou come p edic ions. To ou knowl-
edge, ew such compa a i e da a a e a ailable.
Me hods
Pa ien s
The clinical da a and b eas umou issue samples we e col-
lec ed wi hin he FinHe ial (iden i ie ISRCTN76560285),
whe e 1010 women wi h axilla y node–posi i e o high- isk
axilla y node-nega i e b eas cance we e andomly assigned
be ween Oc obe 2000 and Sep embe 2003 o ecei e ei he
h ee cycles o doce axel ollowed by h ee cycles o luo-
ou acil, epi ubicin and cyclophosphamide (FEC) o h ee
cycles o ino elbine ollowed by h ee cycles o FEC [6,7].
B eas umou e bB2 (HER2) copy numbe s we e de e -
mined cen ally by ch omogenic in si u hyb idiza ion
(CISH), and women wi h HER2-posi i e cance (n=232)
had a second andomisa ion be ween nine weekly in usions
o as uzumab, gi en concomi an ly wi h ei he doce axel
o ino elbine, and simila chemo he apy wi hou as uzu-
mab. A e a median ollow–up ime o 62 mon hs since
andomisa ion, women assigned o doce axel had be e
dis an disease- ee su i al (DDFS, he p ima y objec i e)
han hose assigned o ino elbine (HR 0.66, 95 % CI
0.49–0.91; P=0.010) [6]. The absolu e bene i in 5-yea
DDFS in a ou o he doce axel plus FEC egimen was
5.2 % (86.8 s 81.6 %), and 3.3 % (92.6 s 89.3 %) o
o e all su i al (OS) ac oss all biological sub ypes [6].
3
Labo a o y o Cance Biology, Ins i u e o Medical
Technology, Tampe e, Finland
4
Depa men o Pa hology, HUSLAB, Helsinki Uni e si y
Hospi al and Helsinki Uni e si y, Helsinki, Finland
5
Depa men o Oncology, Tampe e Uni e si y Hospi al and
Uni e si y o Tampe e, Tampe e, Finland
6
Cance Cen e , Kuopio Uni e si y Hospi al, Kuopio, Finland
7
Depa men o Oncology and Radio he apy, Oulu Uni e si y
Hospi al, Oulu, Finland
8
Depa men o Oncology, Tu ku Uni e si y Hospi al, Tu ku,
Finland
9
BioNTech Diagnos ics GmbH, Mainz, Ge many
10
Acomed S a is ik, Leipzig, Ge many
11
Ins i u e o Pa hology a he S -Elisabe h-Hospi al, Cologne,
Ge many
12
Depa men o Oncology, Helsinki Uni e si y Hospi al and
Uni e si y o Helsinki, Helsinki, Finland
438 B eas Cance Res T ea (2016) 157:437–446
123
Immunohis ochemis y
Immunos aining o ER, PgR, HER2 and Ki-67 was
pe o med on issue sec ions cu om o malin- ixed,
pa a in-embedded (FFPE) umou issue a he local
pa hology labo a o ies o he 17 s udy si es (all loca ed in
Finland) acco ding o each labo a o y’s s anda d
p ocedu es.
ER and PgR we e conside ed posi i e when 10 % o
mo e o he cance cells s ained posi i ely. Ki-67 assays
we e analysed by es ima ing he p opo ion o posi i ely
s aining cance cell nuclei ou o all cance cell nuclei in
he issue sec ion, and he esul was p o ided as a pe -
cen age anging om 0 o 100 %. Fo he p esen s udy,
Ki-67 exp ession was conside ed posi i e when C20 % o
cance cell nuclei s ained posi i ely. Local pa hologis s
in e p e ed he ER, PgR and Ki-67 immunos aining esul s,
as pe each ins i u e’s s anda d p ac ice.
Ch omogenic in si u hyb idiza ion (CISH)
Tumou s wi h a sco e o 2?o 3?(on a scale o 0 o 3?)
o HER2 exp ession in IHC we e u he analysed o
HER2 gene ampli ica ion by CISH in one o wo cen al
labo a o ies. The HER2 s a us was conside ed posi i e
when six o mo e gene copies pe nucleus we e p esen . As
in he o iginal ial [6,7], in he p esen s udy, cance
HER2 s a us was conside ed posi i e whene e CISH o
HER2 was posi i e, and nega i e whene e CISH was
nega i e, ega dless o he deg ee o HER2 p o ein
exp ession in IHC.
RT-qPCR
A e pa hologic con i ma ion o ep esen a i eness o he
issue sec ions o p esence o cance , a single whole- ace
10-lm- hick slice om each FFPE umou block was
p ocessed wi h he RNX ac
Ò
RNA ex ac ion ki (BioN-
Tech Diagnos ics GmbH, Mainz) using a magne ic pa i-
cle-based assay (Supplemen al ile 1A). RT-qPCR was
done wi h he MammaType
Ò
ki (BioNTech Diagnos ics
GmbH, Mainz) o ESR1, PGR, ERBB2 and MKI67, and
he wo e e ence genes B2 M and CALM2 on a Ve san
kPCR sys em (Siemens, E langen, Ge many) by applying
one cycle o p ime -speci ic e e se ansc ip ion ollowed
by 40 cycles o nucleic acid ampli ica ion (Supplemen al
ile 1B). The median quan i ica ion cycle (Cq) o each o
he ou genes o in e es (GOI) we e no malized agains
he wo e e ence genes (REF) and p esen ed as DDCq
alues ela i e o he posi i e con ol, ob ained a e sub-
ac ing he DCq alue o he posi i e con ol (pc) om he
DCq o he sample (s) by he o mula
40 DDCq GOIðÞ
S¼40 Cq GOI½
S- meanCq REF½
S

Cq GOI½
pc- meanCq REF½
pc

:
To exclude a majo in luence o a a ying umou cell
con en o he assay esul s, sensi i i y s udies we e
unde aken simila ly as p e iously epo ed [8]. A se ies o
ex eme cases wi h low con en o in asi e ca cinoma and
a ying amoun o DCIS we e analysed be o e and a e
mac odissec ion and i could be con i med ha he TCC did
no in luence he inal es esul [9, Laible e al. submi ed].
The e o e, a majo in luence o TCC on MKI67 mRNA
exp ession can be excluded. Cu -o s o he ma ke s
ERBB2,ESR1 and PGR we e de ined in an independen
echnical coho based on e e ence pa hology IHC esul s.
P ognos ic and p edic i e alue o MKI67 cu -o s had
p e iously been analysed by es ing objec i e cu -o s in
562 A yme ix U133 A da ase s om b eas cance pa ien
coho s ha ing ecei ed ei he no sys emic he apy, only
endoc ine ea men o chemo-endoc ine egimen [10]. In
iew o hese analyses, he MKI67 cu -o was se a he 3 d
qua ile o he no mally dis ibu ed MKI67 exp ession da a
om 90 FFPE b eas cance e e ence umou samples and
hus ough o e lec a co ela e o he s anda d Ki-67 cu -
o a 20 % posi i ely s ained nuclei.
De ini ion o b eas cance biological sub ypes
A e de ining each o he ou bioma ke s ei he posi i e o
nega i e, he molecula sub ype o each umou was de e -
mined using a sligh ly modi ied e sion o he cu en ly
p oposed IHC-based b eas cance molecula sub yping
algo i hm [1] (Supplemen al File 1C). In b ie , luminal A
cance s we e de ined as ha ing high ESR1 and/o PGR
mRNA con en and low ERBB2 and MKI67 con en . Luminal
B cance s we e de ined as ha ing high cance ESR1 and
MKI67 con en , o high ESR1 con en bu low PGR and
ERBB2 con en . Cance s wi h a high ERBB2 mRNA con en
we e conside ed as HER2-posi i e cance s and we e no
u he ca ego ized in o luminal and non-luminal (‘‘en-
iched’’) lesions. T iple-nega i e cance s consis ed o can-
ce s ha had low ESR1,PGR and ERBB2 mRNA con en
i espec i e o cance MKI67 mRNA con en .
The same scheme was used o ca ego ize he cance s
acco ding o he IHC and CISH esul s, bu using p o ein
exp ession (a IHC) and he numbe o HER2 gene copies
(a CISH) in place o cance mRNA con en . Fo example,
cance s ha we e posi i e o ER and PgR (wi h C10 % o
he nuclei ha we e posi i e in each s aining), HER2
nega i e (by CISH) and had low Ki-67 ( 20 % o nuclei
s ained posi i ely a IHC) we e conside ed luminal A
cance s.
B eas Cance Res T ea (2016) 157:437–446 439
123
S a is ical me hods
The esul s we e analysed acco ding o a s a is ical analysis
plan w i en and app o ed p io o he ini ia ion o he
s udy, and he RT-qPCR esul s we e in e p e ed blinded o
he clinical in o ma ion. Kappa (j) s a is ic nume ic alues
a e ca ego ized in o poo (B0.2), ai ([0.2–0.4), mode a e
([0.4–0.6), good ([0.6–0.8) and e y good ([0.8) associ-
a ions, and we e used as a measu e o posi i e pe cen
ag eemen (PPA), nega i e pe cen ag eemen (NPA) and
o e all pe cen ag eemen (OPA). The es s a e accompa-
nied by hei espec i e 95 % con idence in e als (95 %
CI). A wo-sided P alue 0.05 was conside ed signi ican .
The p ima y clinical endpoin was DDFS, de ined as he
ime pe iod be ween he da e o andomisa ion and he da e
o i s dis an me as asis o he da e o dea h when dea h
p eceded de ec ion o dis an ecu ence. O e all su i al
(OS) was de ined as he ime pe iod be ween he da e o
andomisa ion and he da e o dea h. Su i al was analysed
using he Kaplan–Meie me hod.
Uni a iable and mul i a iable Cox p opo ional haza ds
models we e cons uc ed o compa e p ognosis be ween
g oups and o s udy he in e ac ions be ween a iables.
Haza d a ios (HRs) we e calcula ed using a uni a iable
Cox model. In mul i a iable Cox models, a backwa d
selec ion p ocedu e was used o adjus o he co a iables.
Resul s
Pa ien s
An RT-qPCR assay o ESR1,PGR,ERBB2 and MKI67 was
success ully pe o med om b eas cance issues o 769
(76 %) ou o he 1010 pa ien s en e ed o he FinHe ial.
In he emaining 241 cases, cance issues we e no a ail-
able, and he issue block did no consis mos ly o cance
cells, o RNA ex ac ion did no yield good-quali y mRNA.
We included in his s udy all 719 (71 % ou o 1010) cases
wi h success ul RT-qPCR assay o he ou genes and wi h
IHC da a a ailable o sub yping. The inaccessibili y a e
o he issue samples was simila ac oss he s udy ea men
a ms (a modi ied CONSORT diag am shown in Supple-
men al ile 1D). The cha ac e is ics o he pa ien s and
umou s included in he p esen s udy (Table 1) we e
simila o hose o he en i e FinHe ial coho [7].
The median age o he pa ien s a s udy en y was 50.9
yea s ( ange, 25.5–65.8). Tumou s had a mean diame e o
26 mm ±16 mm (6–150 mm), and he majo i y (n=637,
88.6 %) had gi en ise o egional lymph node me as ases
a he ime o he diagnosis. The e we e 511 (71.1 %) ER-
posi i e, 395 (54.9 %) PgR-posi i e and 163 (22.7 %)
HER2-posi i e cance s. A e andom alloca ion, 357
(49.7 %) pa ien s we e ea ed wi h doce axel plus FEC,
362 (50.4 %) wi h ino elbine plus FEC and 83 (50.9 %)
o he 163 pa ien s wi h HER2-posi i e cance ecei ed
as uzumab. The median ollow-up ime a e andomisa-
ion was 62 mon hs, du ing which ime pe iod 112 pa ien s
had dis an cance ecu ence and 62 died.
Conco dance be ween mRNA and IHC assays
Tumou ESR1,PGR and ERBB2 mRNA con en assessed by
RT-qPCR and he co esponding p o ein exp essions
Table 1 Pa ien demog aphics, clinicopa hological da a and e-
quencies o ma ke bina y ca ego ies
N(%)
pT
N=719
1 290 (40.33)
2 369 (51.32)
3 47 (6.54)
4 13 (1.81)
pN
N=718
0 80 (11.14)
1 621 (86.49)
2 16 (2.23)
3 1 (0.14)
His ological ype
N=719
Duc al 575 (79.97)
Lobula 131 (18.22)
Papilla y 2 (0.28)
Mucinous 2 (0.28)
Medulla y 9 (1.25)
His ological g ade
N=694
I 102 (14.70)
II 287 (41.35)
III 305 (43.95)
Adju an chemo he apy
N=719
Doce axel 357 (49.65)
Vino elbine 362 (50.35)
HER2-pos
N=163
T as uzumab 83 (50.92)
No T as uzumab 80 (49.08)
Type o Su ge y
N=719
To al Mas ec omy 430 (59.81)
B eas Conse ing 289 (40.19)
440 B eas Cance Res T ea (2016) 157:437–446
123
de e mined by IHC o ER and PgR and DNA ampli ica ion
s a us by CISH o HER2 showed good conco dance,
whe eas cance MKI67 mRNA con en and p o ein exp es-
sion co ela ed only mode a ely well (Table 2). Many o he
disco dan cases be ween IHC and he mRNA assay had a
high cance MKI67 mRNA con en , bu despi e his, 20 %
o cance cell nuclei s ained posi i ely wi h IHC (Fig. 1).
P ognos ic alue o cance MKI67 mRNA con en
and Ki-67 exp ession
Pa ien s wi h low b eas umou MKI67 mRNA con en o
low ( 20 %) Ki-67 exp ession had mo e a ou able DDFS
and OS as compa ed o hose wi h high MKI67 mRNA
con en o Ki-67 exp ession. Each me hod p oduced
oughly simila haza d a ios o DDFS and OS (Fig. 2).
In a mul i a ia e Cox eg ession analysis whe e he ype
o chemo he apy ( ino elbine-FEC o doce axel-FEC), he
axilla y nodal s a us (pN0, pN1, pN2 o pN3), umou size
(as a con inuous a iable), his ological g ade (as a con-
inuous a iable) and cance MKI67 mRNA con en (as a
con inuous a iable) we e en e ed as co a iables, low
umou MKI67 mRNA con en was independen ly associ-
a ed wi h a ou able DDFS (adjus ed HR 0.51; 95 % CI,
0.29–0.90; P=0.019) oge he wi h a nega i e axilla y
nodal s a us (P 0.0001) and small cance size
(P=0.006). A low cance MKI67 mRNA con en was also
independen ly associa ed wi h a ou able OS (adjus ed HR
0.44; 95 % CI, 0.23-0.87; P=0.018) in addi ion o he
axilla y nodal s a us (P=0.003) and umou size
(P=0.006). When Ki-67 p o ein exp ession was en e ed
in o he same models in place o cance mRNA con en ,
Ki-67 was no signi ican ly associa ed wi h DDFS
(P=0.266), bu when OS was selec ed as he endpoin ,
low cance Ki-67 exp ession was associa ed wi h a ou -
able su i al (adjus ed HR 0.43; 95 % CI, 0.24–0.77;
P=0.005) oge he wi h he axilla y nodal s a us
(P=0.002) and small umou size (P=0.006).
Conco dance o molecula sub yping wi h IHC
and RT-qPCR
The me hod o Ki-67 assessmen had subs an ial impac on
making he dis inc ion be ween luminal A and B cance s.
O he 189 cance s ha we e classi ied as luminal A by
IHC/CISH, only 102 (54.0 %) we e simila ly classi ied,
when MKI67 mRNA exp ession was used in place o Ki-67
p o ein s aining wi h he 87 disco dan cases being classi-
ied as ei he luminal B (n=75, 39.7 %) o HER2 posi i e
(n=12, 6.4 %, Table 3). O he 251 cance s ha we e
classi ied as luminal B by IHC/CISH, 180 (71.7 %) we e
simila ly classi ied using MKI67 mRNA exp ession, 48
(19.1 %) we e classi ied as luminal A, 17 (6.8 %) as HER2
posi i e and 6 (2.4 %) as iple nega i e. O he 156 and
294 umou s classi ied as luminal A and luminal B by RT-
Table 2 Ag eemen be ween
RT-qPCR-based and IHC-based
bioma ke assessmen s
ESR1 PGR ERBB2 MKI67
Conco dance 660/719 (91.8 %) 593/719 (82.5 %) 660/719 (91.8 %) 516/688 (75.0 %)
PPA 490/511 (95.9 %) 368/395 (93.2 %) 140/163 (85.9 %) 369/414 (89.1 %)
NPA 170/208 (81.7 %) 225/324(69.4 %) 520/556 (93.5 %) 147/274 (53.7 %)
Kappa s a is ic 0.80 (0.75–0.85) 0.64 (0.58–0.70) 0.77 (0.72–0.83) 0.45 (0.38–0.52)
P 0.0001 P 0.0001 P 0.0001 P 0.0001
PPA Posi i e pe cen ag eemen , NPA Nega i e pe cen ag eemen
Fig. 1 A sca e plo depic ing he ela ion be ween umou MKI67
mRNA con en measu ed wi h RT-qPCR, and Ki-67 exp ession
de e mined by immunohis ochemis y (IHC). Ve ical axis, umou
Ki-67 exp ession (IHC, %); ho izon al axis, umou ela i e MKI67
mRNA exp ession. The cu -o o posi i i y was 20 % in he Ki-67
p o ein assays ( he ho izon al line) and 34.8 in he MKI67 mRNA
(qPCR) assays ( he e ical line). Sec ions Aand Ddepic he
disco dan cases, and sec ions Band Cdepic he conco dan cases
B eas Cance Res T ea (2016) 157:437–446 441
123

qPCR, espec i ely, 102 (65.4 %) and 180 (61.2 %) we e
classi ied as luminal A o B also wi h IHC/CISH.
In luence o cance MKI67 mRNA exp ession-based
and Ki-67 p o ein exp ession-based sub ypes
on ou come
The e was no signi ican di e ence in DDFS o OS
be ween pa ien s ea ed wi h adju an doce axel plus FEC
and hose ea ed wi h ino elbine and FEC in he subse s
wi h luminal A, HER2-posi i e o iple-nega i e b eas
cance when each sub ype was de ined ei he wi h IHC/
CISH o wi h RT-qPCR (DDFS and OS s a is ics o each
sub ype acco ding o chemo he apy agen shown in Sup-
plemen al ile 1E). In e es ingly, when luminal B sub ype
was de ined by MKI67 mRNA exp ession, pa ien s ea ed
wi h doce axel plus FEC had signi ican ly mo e a ou able
DDFS and OS as compa ed wi h hose ea ed wi h
ino elbine plus FEC ( o DDFS, HR 0.52, 95 % CI
0.29–0.94, P=0.031; OS, HR 0.24, 95 % CI 0.09–0.65,
P=0.005). In con as no signi ican di e ence in DDFS
o OS was ound, when he luminal B sub ype was de ined
Fig. 2 In luence o cance MKI67 mRNA exp ession and Ki-67
p o ein exp ession on DDFS (panels aand c) and su i al (panels
band d). Resul s ob ained by measu ing MKI67 mRNA exp ession
a e shown in panels aand b, and hose ob ained by assessing Ki-67
p o ein exp ession in panels cand d
Table 3 Conco dance o b eas
cance sub ypes when cance
Ki-67 exp ession is assessed
wi h IHC and MKI67 mRNA
exp ession wi h RT-qPCR
RT-qPCR-based
Luminal A Luminal B HER2 pos TNBC To al
N(%) N(%) N(%) N(%) N(%)
IHC-based
Luminal A 102 65.4 75 25.5 12 6.8 0 0.0 189 26.3
Luminal B 48 30.8 180 61.2 17 9.7 6 6.5 251 34.9
HER2 pos 5 3.2 12 4.1 140 79.6 6 6.5 163 22.7
TNBC 1 0.6 27 9.2 7 4.0 81 87.1 116 16.1
To al 156 100.0 294 100.0 176 100.0 93 100.0 719 100.0
442 B eas Cance Res T ea (2016) 157:437–446
123
wi h Ki-67 p o ein exp ession (P[0.10 o bo h analyses;
Fig. 3).
The ype o adju an chemo he apy ( es ed doce axel
plus FEC s ino elbine plus FEC) had an independen
in luence on DDFS in he subse o pa ien s who had
luminal B cance de ined by cance MKI67 mRNA con en
in a mul i a iable analysis (HR 0.44; 95 % CI 0.23–0.84,
P=0.013), oge he wi h cance his ological g ade ( es ed
as a con inuous a iable; HR 1.67, 95 % CI 1.03–2.72,
P=0.039) and umou size ( es ed as a con inuous ac o ;
HR 1.02, 95 % CI 1.00–1.04, P=0.026). Simila ly, when
OS was used as he end poin in place o DDFS and he
luminal B sub ype was de ined by cance MKI67 mRNA
con en , doce axel-con aining chemo he apy was indepen-
den ly associa ed wi h a ou able su i al (HR 0.22; 95 %
CI 0.08–0.60, P=0.003) oge he wi h his ological g ade
(HR 2.29, 95 % CI 1.15–4.57; P=0.019), while umou
size los i s signi icance. Unlike MKI67 mRNA con en , Ki-
67 p o ein exp ession did no ha e independen in luence
on DDFS o OS in hese models. When he luminal B
sub ype was de ined wi h umou MKI67 mRNA con en ,
he in e ac ion wi h he ype o adju an chemo he apy
gi en was signi ican (P=0.040) when OS was selec ed
as he end poin , bu no when DDFS was conside ed
(P=0.352). No in e ac ion wi h ei he OS o DDFS and
he ype o adju an chemo he apy was p esen when he
luminal sub ype was de ined wi h Ki-67 p o ein exp ession
(P=0.658 and 0.699, espec i ely).
Discussion
We app oxima ed commonly used b eas cance biologi-
cal sub ypes using RT-qPCR and compa ed he esul s
wi h he sub ypes de ined by IHC (and wi h CISH o
de ec HER2 ampli ica ion) wi hin he amewo k o a
la ge andomized clinical ial. The sub ypes de ined wi h
each me hod ag eed mode a ely well wi h mos disc ep-
ancy occu ing in he luminal B sub ype. Bo h high
cance Ki-67 p o ein exp ession and high MKI67 mRNA
con en we e associa ed wi h un a ou able DDFS and OS
in a uni a iable analysis wi h app oxima ely simila
haza d a ios, bu only umou MKI67 mRNA con en
emained signi ican in a mul i a iable model o DDFS
when bo h pa ame e s we e en e ed in o he same model
a e a s epwise selec ion p ocess o he co a iables such
as umou size, nodal s a us, his ological g ade and he
ype o ea men gi en.
Fig. 3 Dis an me as asis- ee su i al (panels aand b) and o e all
su i al (panels cand d) o pa ien s ea ed wi h adju an doce axel
plus FEC and hose ea ed wi h ino elbine plus FEC in he subse o
pa ien s wi h luminal B b eas cance . Panels aand c, he luminal B
sub ype was de ined wi h MKI67 mRNA exp ession; panels band d,
he luminal B sub ype was de ined wi h Ki-67 p o ein exp ession
B eas Cance Res T ea (2016) 157:437–446 443
123
A key di e ence be ween luminal A and luminal B
sub ypes is a highe cell p oli e a ion a e in he la e ,
which is o en assessed by es ima ing he p opo ion o
cance cells ha s ain posi i ely o Ki-67 a e immuno-
his ochemical s aining. In e es ingly, when he luminal B
ype was de ined using cance MKI67 mRNA con en in
place o Ki-67 exp ession assessed wi h immunohis o-
chemis y, pa ien s wi h luminal B b eas cance we e
ound o bene i mo e om adju an doce axel plus FEC
han om adju an ino elbine plus FEC, which associa-
ion could no be de ec ed when he luminal B b eas
cance sub ype was de ined by Ki-67 p o ein exp ession
wi h immunohis ochemical s aining.
Biological sub yping o b eas cance is he basis o
selec ion o sys emic cance ea men [1]. O he ou
bioma ke s commonly used o his pu pose, i.e. ER, PgR,
HER2 and Ki-67, he assays o Ki-67 ha e u ned ou he
mos challenging ones o s anda dize and o make ep o-
ducible. Fo example, in a s udy ca ied ou in a ew leading
pa hology labo a o ies, he e was subs an ial a iabili y
be ween he labo a o ies in sco ing o Ki-67 exp ession om
sha ed b eas cance issue slides s ained wi h IHC, and
a emp s o educe he in e labo a o y a iabili y we e only
pa ially success ul [4]. In he p esen s udy, IHC s aining o
Ki-67 was done locally in many pa hology labo a o ies using
he ins i u ional s aining p o ocols and was assessed by many
pa hologis s, whe eas cance MKI67 mRNA con en was
de e mined cen ally in one labo a o y. To educe he
po en ial a iabili y in Ki-67 s aining and sco ing, we con-
side ed ca ying ou s aining o Ki-67 also cen ally, bu due
o he di icul ies o s anda dize Ki-67 immunos aining e en
in leading labo a o ies and o es ablish a e e ence p ocedu e
[4], we p e e ed o use he Ki-67 s aining esul s epo ed
o iginally by he local labo a o ies om whole umou issue
sec ions as he compa a o o he MKI67 mRNA assay.
Image analysis o Ki-67 om IHC s ained slides is a
p omising me hod o imp o e he ep oducibili y o Ki-67
sco ing om immunos ained slides, bu , o ou knowledge,
no s anda d pa ame e alues o sco ing o he nuclei as
ei he posi i e o nega i e a e a ailable. To es ima e how
well he locally assessed Ki-67 assays done om whole
umou issue sec ions migh co ela e wi h a cen ally done
Ki-67 assay, we analysed cance Ki-67 exp ession om
TMAs (as whole umou sec ions we e no a ailable) con-
aining issue om 745 b eas cance s using image analysis
[11]. The median cance Ki-67 exp ession u ned ou o be
simila wi h image analysis and locally done IHC (19.7 and
20.0 %, espec i ely), and he wo assays showed s ong
co ela ion (P 0.0001, Spea man’s ho 0.633). These
obse a ions sugges ha cen ally done image analysis o
Ki-67 migh ha e esul ed in simila conclusions had i been
selec ed as he compa a o assay in place o he local Ki-67
IHC assays.
The sub ypes de ined wi h MKI67 mRNA we e associ-
a ed wi h su i al ou comes ha ag ee well wi h he esul s
ob ained wi h IHC om o he clinical ials [9,10,12–14].
Pa ien s wi h he luminal A sub ype had he bes 5-yea
DDFS, pa ien s wi h HER2 posi i e and iple-nega i e
cance had he leas a ou able ou comes, while pa ien s
wi h luminal B cance had an ou come in e media e o
hese sub ypes (see Supplemen File 1E). These esul s a e
well in ag eemen despi e sligh dissimila i ies in he de -
ini ion o luminal B and HER2-posi i e sub ypes be ween
he ials.
Taxane-con aining adju an egimens a e e ec i e in
he ea men o ea ly b eas cance bu a e associa ed wi h
side e ec s, and he e o e, me hods o op imize pa ien
selec ion o egimens ha con ain a axane a e needed.
The cu en inding ha pa ien s wi h luminal B cance
ha e longe DDFS and OS when ea ed wi h doce axel
plus FEC as compa ed wi h ino elbine plus FEC is sup-
po ed by obse a ions made by Jacquemie e al. and Ni z
e al. who ound ha chemo he apy con aining doce axel
was associa ed wi h a signi ican educ ion in he isk o
elapse in he subse o pa ien s wi h luminal B b eas
cance in he PACS 01 ial [13] and WSG-AGO EC-Doc
ial [12], espec i ely. Bo h o hese ials compa ed
doce axel-con aining egimens wi h s anda d an h acy-
cline-con aining ea men s. In he BCIRG 001 ial ha
compa ed doce axel, doxo ubicin and cyclophosphamide
(TAC) e sus luo ou acil, doxo ubicin and cyclophos-
phamide (FAC) in he ea men o ope able node-posi i e
b eas cance , only pa ien s wi h ER-posi i e umou s wi h
ei he high Ki-67 exp ession o HER2 o e exp ession had a
s a is ically signi ican imp o emen in disease- ee su -
i al when ea ed wi h TAC [14]. Howe e , unlike hese
s udies, we did no ind a su i al bene i om he doc-
e axel-con aining egimen in he subse o women wi h
HER2 posi i e cance . In FinHe , hal o he pa ien s wi h
HER2-ampli ied cance we e andomly assigned o ecei e
adju an as uzumab, which may ha e masked he
po en ial doce axel bene i in his sub ype and may ha e
educed he s a is ical powe o de ec he associa ion.
The PAM50 gene exp ession a ay has also been e al-
ua ed in p edic ing he po en ial bene i o adding a axane
o an h acycline-based chemo he apy, bu none o he
PAM50-de i ed sub ypes including he luminal B sub ype
we e p edic i e o a axane bene i in he GEICAM/9966
and he NCIC CTG MA.21 andomized phase III ials [15,
16]. Simila ly he Endop edic gene exp ession assay did
no p edic axane bene i in he GEICAM/9966 s udy
popula ion [17].
The limi a ions o he s udy include he e ospec i e
na u e o he s udy, al hough we de e mined umou MKI67
mRNA wi hou knowledge o he clinical da a and planned
he s a is ical analyses p ospec i ely. We es ed he
444 B eas Cance Res T ea (2016) 157:437–446
123
me hods wi hin he con ex o a ela i ely la ge andomized
ial bu lacked a alida ion se ies, and some subg oup
analyses ha e limi ed powe . Howe e , he PCR me hod
used u ned ou o be ep oducible ac oss mul iple es ing
si es o all ou bioma ke s including MKI67 mRNA
(Laible e al., manusc ip submi ed o publica ion). The
de ails o he IHC me hods used o assaying Ki-67 in he
local pa hology labo a o ies we e no cap u ed du ing he
FinHe ial, as Ki-67 was no a p o ocol-manda ed assay,
bu mos pa hology labo a o ies in Finland assess Ki-67
om he umou ho spo a eas. The ecommended cu -o
o ER and PgR posi i i y is now 1 % and no longe 10 %
as i was a he ime when he FinHe ial acc ued pa ien s,
bu he p opo ion o b eas cance s whe e ER o PgR a e
exp essed in 1 % o 10 % o nuclei is small [18].
Conclusions
Measu ing o cance ESR1,PGR and ERBB2 mRNA co -
ela ed well wi h he esul s ob ained wi h IHC and CISH
in clinical pa hology labo a o ies. Tumou MKI67 mRNA
con en quan i a ed wi h RT-qPCR is associa ed wi h
DDFS and OS o pa ien s ea ed wi h mode n adju an
egimens. The esul s sugges ha assessmen o umou
MKI67 mRNA con en may be aluable o selec ion o
pa ien s o doce axel-con aining adju an he apy. Since
he immunohis ochemical assay esul s o Ki-67 exp es-
sion a e challenging o ans e be ween labo a o ies, and
he assay o measu ing cance MKI67 mRNA con en wi h
RT-qPCR migh be less challenging o s anda dize han
IHC s ainings, pe o ming s udies ha e alua e in e labo-
a o y compa isons o cance ESR1,PGR,ERBB2 and
MKI67 mRNA con en using RT-qPCR a e wa an ed.
Acknowledgmen s We hank Elke Vel up, Susanne Scha , Silke
Claas and To s en Ach o excellen echnical suppo in de eloping
molecula sub yping echnologies, and. D s. Thomas Kelle and
S e an Webe o pe o ming s a is ical analyses. The s udy was
suppo ed inancially by he Academy o Finland, Cance Socie y o
Finland, Jane and Aa os E kko Founda ion, he Sig id Juselius
Founda ion, and he Resea ch Funds o he Helsinki Uni e si y
Hospi al.
Au ho con ibu ions HJ, RMW and US we e in ol ed in he
concep ion o he s udy. RMW pe o med he RT-qPCR assays; HS,
JI, PH, P-LK-L, PA, TT-H, SJ and HJ p o ided s udy da a and
ma e ials; SW pe o med he s a is ical analysis and w o e he s a-
is ical plan; RMW, HJ, SL, KS, ML, SE and US in e p e ed he da a;
RMW, SL and HJ d a ed he manusc ip ; all au ho s ead and
app o ed he inal manusc ip .
Compliance wi h e hical s anda ds
Con lic o in e es s RM Wi z is he ounde and CEO o STRA-
TIFYER Molecula Pa hology GmbH. S Eid has s ock op ions in
STRATIFYER Molecula Pa hology GmbH. S Lakis is an employee
o STRATIFYER Molecula Pa hology GmbH. U Sahin is he oun-
de and CEO o BioNTech Diagnos ics GmbH. K Schlombs and M
Laible a e employees o BioNTech Diagnos ics GmbH. No po en ial
con lic s o in e es s we e disclosed by he o he au ho s.
E hical s anda ds Each s udy pa icipan p o ided signed in o med
consen o he clinical ial, and a sepa a e consen o use umou
issue o esea ch ela ed o he FinHe s udy. An E hics Commi ee
o he Helsinki Uni e si y Hospi al app o ed he p o ocol o he
cu en s udy (HUS 124/13/03/02/2014, pe mission g an ed on Ap 9,
2014).
Open Access This a icle is dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion-NonComme cial 4.0 In e na ional
License (h p://c ea i ecommons.o g/licenses/by-nc/4.0/), which pe -
mi s any noncomme cial use, dis ibu ion, and ep oduc ion in any
medium, p o ided you gi e app op ia e c edi o he o iginal
au ho (s) and he sou ce, p o ide a link o he C ea i e Commons
license, and indica e i changes we e made.
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