PRECLINICAL STUDY

Prognostic and biological significance of proliferation and HER2expression in the luminal class of breast cancer

Dena A. Jerjees • M. Alabdullah • Andrew R. Green •

Alaa Alshareeda • R. D. Macmillan •

Ian O. Ellis • Emad A. Rakha

Received: 16 January 2014 / Accepted: 26 March 2014 / Published online: 18 April 2014

� Springer Science+Business Media New York 2014

Abstract The definition of Luminal-B subclass of breast

cancer (BC) varies in literature. In this study, we have

compared the proliferation status; assessed using KI67

labeling index (KI67-LI), and HER2-expression in estro-

gen receptor positive (ER?) BC to assess their impact on

the biological and clinical characteristics of luminal-BC.

1547 (73.8 %) well-characterized clinically annotated

stage I–III ER ? BC were assessed for expression of KI67,

HER2 (ASCO guidelines), and a large panel of relevant

biomarkers (no = 37). 46.3 % of the cases show high

KI67-LI ([13 %) and 8.4 % show HER2? and both

markers are positively associated with younger age, higher

tumor grade and poorer outcome. High KI67-LI and

HER2? are associated with upregulation of ER-coactiva-

tors and proliferation-related markers and with downregu-

lation of good prognostic markers. High KI67-LI is

associated with larger size, advanced stage, and lympho-

vascular invasion (LVI) and with downregulation of

luminal-enriched and DNA-damage repair markers. In

contrast, HER2? is associated with upregulation of ER-

regulated proteins and E-cadherin. When analysis is

restricted to high KI67-LI subgroup, HER2? shows an

association with upregulation of differentiation-associated

proteins and E-cadherin. Conversely, within HER2? class,

high KI67-LI maintains its association with downregula-

tion of differentiation-associated/luminal-enriched pro-

teins. Outcome analyses indicate that both markers are

independently associated with shorter survival but HER2?

is associated with a worse outcome. Although both are

associated with high proliferation and poor prognosis

within ER ? BC, HER2? is less frequent than high KI67-

LI. Unlike KI67, HER2 seems to independently drive the

aggressive behavior of ER? tumors without downregula-

tion of luminal proteins.

Keywords Breast carcinoma � Molecular classes �Immunohistochemistry � HER2 � KI67

Introduction

In mammary tissue, estrogen receptor (ER) regulates

growth and development by regulating the balance between

cell proliferation and differentiation [1, 2]. In BC, ER

expression which is observed in 80–85 % of cases, pro-

vides a prognostic and predictive value; indicating good

prognosis [3] and response to hormone therapy [4]. Gene

expression profile (GEP) has classified BC into distinct

molecular classes and identified ER in addition to HER2

and proliferation as key players regulating its molecular

profile. ER-expressing tumors are clustered together in the

so called ‘‘luminal class,’’ which is associated with a good

prognosis [5]. Subsequent GEP studies have sub-stratified

luminal-BC into at least two subclasses: A and B with the

latter associated with poor prognosis [6–8]. Although most

immunohistochemical studies have defined luminal-A

D. A. Jerjees (&) � A. R. Green � A. Alshareeda �I. O. Ellis � E. A. Rakha

Department of Histopathology, University of Nottingham and

Nottingham University Hospitals NHS Trust, Nottingham, UK

e-mail: denaakramj@yahoo.co.uk

D. A. Jerjees

Department of Pathology, Mosul School of Medicine, Mosul,

Iraq

M. Alabdullah

Department of Surgery, School of Medicine, Mosul, Iraq

R. D. Macmillan

Breast Institute, City Hospital, Nottingham, UK

123

Breast Cancer Res Treat (2014) 145:317–330

DOI 10.1007/s10549-014-2941-7

tumors as hormone receptor-positive and HER2-negative,

the definition of luminal-B class remains imprecise. Some

studies have used HER2 positivity [9–11] while others used

KI67-LI [12] and some have used combined HER2-posi-

tivity and/or high proliferation to define luminal-B tumors

[13]. Others contest this and place all HER2-positive

tumors, regardless of hormone receptor expression in the

HER2-positive subgroup [14]. Cheang et al. [15] using

qRT-PCR gene expression profiles have classified BC into

luminal-A (28 %) and luminal-B (19 %). Although in their

study the expression profiling classified hormone receptor-

positive/HER2-positive tumors as luminal-B, using surro-

gate immunohistochemical markers, these authors sepa-

rated luminal tumors (hormone receptor-positive) into

three subgroups based on HER2 and KI67; (i) luminal-A

class defined as HER2-negative and KI67-negative/low,

(ii) luminal-B as HER2-negative and KI67-high and (iii)

luminal-HER2 positive as HER2-positive regardless of

KI67 expression. Survival analysis showed similar out-

come for tumors classified as luminal-B and luminal-HER2

positive. The role of proliferation in defining the luminal-B

class is highlighted in a recently published statement [12].

Although HER2 is a major determinant of BC molecular

profile, there appears to be interaction between HER2 and

ER pathways and this interaction depends on several fac-

tors including ER level, transcriptional co-repressors, and

co-activators [16]. Proliferation plays an important role in

BC outcome and is associated with biological, molecular,

and clinical features characteristic of aggressive behavior

[17, 18]. Upregulation of proliferation-related genes was

also a common theme in most prognostic gene signatures

[12]. Although an inverse correlation between KI67 and

hormone receptor expression has been documented, cor-

relation between KI67 and HER2 remains unclear and both

positive and negative associations have been reported [19–

21]. Therefore, this study aims to investigate the biological

and clinical significance of KI67 and HER2 expression and

coexpression in a large and well-characterized series of

ER-positive/luminal-BC.

Materials and methods

1547 well-characterized and clinically annotated estrogen

receptor (ER)-positive early stage (I–III) operable BCs were

investigated. These cases are part of the Nottingham pri-

mary breast carcinoma series which is composed originally

of 1900 patients who presented between 1988 and 1998.

This series is well-characterized in terms of uniform man-

agement protocol and patients’ prospective assessment. All

patients were\70; the mean age was 54 years. Information

regarding clinical history, age, and menopausal status,

together with tumor characteristics including tumor

histological grade, tumor type, lymphovascular invasion,

lymph node stage, tumor size, and Nottingham Prognostic

Index (NPI) [22], were all taken into consideration. Patient

management was uniform and was based on tumor charac-

teristics by NPI and hormone receptor status as previously

described [23]. None of the patients in this study received

neoadjuvant therapy or adjuvant targeted HER2 treatment.

In terms of the survival data, all were assessed on a pro-

spective basis. BC-specific survival (BCSS) (mean = 123 -

months) is defined as the time in months from the primary

surgery to patient death due to BC. Death due to other causes

is censored. Distant metastasis free interval is defined as the

period in months between the primary surgery and the

occurrence of distant metastasis (mean = 115 months). This

data is routinely updated on the system together with other

information including, loco-regional recurrence that was used

to assess disease free interval (DFI).

Immunohistochemistry

BC tissue microarray (TMA) and whole BC tissue sections

were stained using Streptavidin Biotin complex process.

Regarding HER2, Rabbit antihuman HER2 protein (Dako,

Denmark) was used as a primary antibody with an incu-

bation period of 45 min. The chromogenic substance used

in this method was 3-30 Diaminobenzidin tetrahydrochlo-

dide (Dako liquid DAB plus, K3468, Dako, Denmark). The

counterstaining used for TMA sections was Mayer’s He-

matoxyllin. Furthermore, the positive control was used in

each run according to the supplier’s information and in

such case, the kidney was used. The same protocol for

KI67, but the primary antibody was mouse monoclonal

antibody against KI67 (clone MIB1; Dako, Denmark),

diluted 1:100 in swine serum and then incubated for

60 min after application to each slide using full-face sec-

tions; the cutoff point used to evaluate the KI67 labeling

index (LI) was [13 as previously defined [24]. A large

panel of antibodies was used in this study; the details are in

Table 1. The procedure was performed according to stan-

dard protocol for each antibody as part of studies per-

formed by our group. Details are the same as previously

published [25–28].

Immunohistochemical scoring

Immunoreactivity of HER2 in TMA cases was scored as

proportion of cells stained using standard Hercept test

guidelines. Only cancer cells localized within tissue cores

were considered. High-resolution digital image (Nano-

ZoomerHamamatsuphotomix, Welwyu Garden City, UK)

at 209 and 40ld9, magnification, using a web-based

318 Breast Cancer Res Treat (2014) 145:317–330

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Table 1 Details of the immunohistochemical markers utilized in this study

Category Markers Dillution Source Pretreatment

1-Oestrogen receptor related proteins

Hormone receptors (HRs) ER [clone 1D5] 1:150 DAKO Microwave

Progesterone receptor(PgR)[clone PgR 636] 1:100 DAKO

Androgen(AR) [clone F39.4.1] 1:30 Biogenex

luminal cytokeratins (CKs) Ck7/8 [clone CAM 5.2] 1:2 Becton Dickinson Microwave

Ck18 [clone DC10] 1:50 DAKO

Ck19 [clone BCK 108] 1:100 DAKO

Basal cytokeratins Ck5/6 [clone D5/16134] 1:100 BoehringerBiochemica Microwave

Ck14 [clone LL002] 1:100 Novocastra

Ck17[clone E3] 1:20 Abcam

Other ER related proteins &

ER co-regulators

Trefoil factor 3(TFF3) 3lg/ml Abcam Microwave

Trefoil factor 1(TFF1 1:2000 Abcam Microwave

Forkhead box protein A1 (FOXA1) [clone 2F83] 1:2,000 Abcam Microwave

AGTR1(angiotensin II receptor type 1)[1E101A9] 1:100 Abcam Microwave

Trans-acting T-cell-specific transcription factor

(GATA3) [HG3-31]

1:80 Santa Cruz

1.30 Biotechnology

Gross cystic disease fluid protein (GCDFP-15) 1:300 Novocastra

Co-activator associated arginine methyl transferase

(CARM1)[clone NB100]

1:100 NOVUS

1:30 NOVUS

Proline, glutamate and leucin rich protein

1(PELP1)[clone NB100]

Abcam

Transferrin receptor (resistance to endocrine treatment: CD

71[clone 10F11]

2-Cell cycle, proliferation and apoptosis related markers

Cell cycle related proteins Cyclin D1 1:50 Neomarkers

Makers of proliferation Thymidine kinase1(TK1)[clone ab72] 5lg/ml Abcam Pressure

Phosphatidylinositol-3 kinase (PI3K) 1:100 Neomarker cook

Protein kinase B (Akt), [clone 17.A] 1:50 DAKO Microwave

Ki-67[clone MIB1] 1:100 DAKO

Apoptosis related markers Bcl2 (pro-apoptotic) [clone 124] 1:100 DAKO Microwave

3-Tumor suppressor proteins, cell adhesion molecules and mucins

Tumor suppressor proteins p53 [clone DO7] 1:50 Novocastra Microwave

BRCA1Ab-1 [clone MS110] 1:150 Oncogene Res Microwave

BEX1[Ab69032] 1:3500 Product Microwave

Fragile histidine triad protein (FHIT) [clone ZR44] 1:600 Abcam

Cell adhesion molecules E-Cadherin [clone HECD-1] Microwave 1:100 Zymed Laboratories Microwave

P-Cadherin [clone 56] 1:200 BD Biosciences

N-Cadherin

Mucins MUC-1core[Ma552] 1:250 Novocastra Microwave

MUC1 1:300 Novocastra Microwave

HER family proteins HER1 [clone EGFR.113] 1:10 Novocastra Microwave

HER2 [clone cerbB-2] 1:250 DAKO Microwave

HER3[clone RTJ1] 1:20 Novocastra

HER4 [clone HFR1] 6:4 Neomarkers

DNA-damage repose genes RAD51 [clone Ab88572] 1:70 Abcam Microwave

BRCA1Ab-1 [clone MS110] 1:150 Calbiochem Microwave

BARD1 [NBP1 19636] 1:50 Novous biological Microwave

yH2AX[ clone Ab 22551] 1:600 Abcam Microwave

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interface (Distiller;slidepatht, Dublin, Ireland) was used to

score the TMAs. In addition, chromogenic in situ hybrid-

ization technique (CISH) is used in borderline cases. Dual

color CISH is considered as an extension of HER2 FISH

PharDXTM protocol. However, the air drying and

mounting steps and ultimate dehydration were not applied.

The details of CISH are as previously published [29]. The

evaluation of the dual color CISH slides was performed

Table 2 The association between HER2 and KI67-LI in ER-positive/

luminal tumors

HER2-negative

(%)

HER2-positive

(%)

X2; p value

Low KI67-LI 620 (94) 41 (6) 41.5, \0.001

High KI67-LI 680 (83) 142 (17)

Total 1300 (88) 183 (12) 1483

Table 3 Association between HER2, KI67-LI, and the clinicopathological variables

Clinicopathological variables HER2- HER2? p value Low KI67-LI High KI67-LI p value

Age (years)

\50 370(29.3%) 52(44.8%) 0.001* 164(27.4%) 197(37.2%) \0.001*

[50 892(70.7%) 64(55.2%) 434(72.6%) 333(62.8%)

Menopausal status

Pre 429(33.4%) 53(45.7%) 0.008* 207(33.7%) 207(38.9%) 0.06

Post 854(66.6%) 63(54.3%) 408(66.3%) 325(61.1%)

Size

\2 CM 708(56.3%) 52(45.2%) 0.02 371(62%) 244(46%) \0.001*

[2 CM 549(43.7%) 63(54.8%) 227(38%) 284(54%)

Tubule

1 108(8.8%) 29(1.8%) 0.001* 80(13.5%) 7(1.4%) \0.001*

2 493(40.1%) 34(29.8%) 255(42.9%) 184(35.9%)

3 628(51.1%) 78(68.4%) 259(43.6%) 322(62.8%)

Pleomorphism

1 49(4.0%) 0(0.0%) \0.001* 32(5.4%) 2(0.4%) \0.001*

2 668(54.4%) 22(19.5%) 396(66.8%) 153(29.8%)

3 5119(41.6%) 919(80.5%) 165(27.8%) 358(69.8%)

Mitosis

1 644(52.4%) 10(8.8%) \0.001* 446(75.1%) 72(14.0%) \0.001*

2 252(20.5%) 25(21.9%) 96(16.2%) 143(27.9%)

3 333(27.1%) 79(69.3%) 52(8.8%) 298(58.1%)

Grade

1 334(26.2%) 4(3.5%) \0.001* 240(39%) 24(4.5%) \0.001*

2 555(43.5%) 23(20.5) 299(48.5%) 176(33.2%)

3 387(30.3%) 88(76.5%) 77(12.5%) 330(62.3%)

Stage

1 1831(65.1%) 62(53.9%) 0.03 422(68.5%) 285(53.9%) \0.001*

2 357(28%) 40(34.8%) 153(24.8%) 196(37.1%)

41(6.7%) 48(9.1%)3 88(6.9%) 13(11.3%)

Vascular invasion

NO 883(70.5%) 72(62.1%) 0.05 452(75.6%) 323(61.2%) \0.001*

Definite 370(29.5%) 44(37.9%) 146(24.4%) 205(38.8%)

NPI*

1 569(44.5%) 12(10.5%) \0.001* 359(58.4%) 100(18.9%) \0.001*

2 567(44.4%) 71(62.3%) 218(35.4%) 312(58.9%)

3 142(11.1%) 31(27.2%) 38(6.2%) 530(100.0%)

* significant, NS not significant (p [ 0.07), (borderline: p = 0.01–0.07) and NPI Nottingham prognostic index

320 Breast Cancer Res Treat (2014) 145:317–330

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Table 4 Association between HER2, KI67-LI, and different proteins used in this study

Markers HER2- HER2? p value Low Ki67 LI High Ki67 LI p value

Hormone receptors

Progesterone receptor

NEG 247(19.8%) 46(40.7%) \0.001* 113(19.2%) 117(22.5%) NS

POS 1001(80.2%) 67(59.3%) 477(80.8%) 402(77.5%)

Androgen receptor

NEG 257(22.4%) 37(35.9%) 0.002* 102(19%) 136(27.9%) 0.001*

POS 892(77.6%) 66(64.1%) 434(81%) 352(72.1%)

Luminal cytokeratins

CK7/8

NEG 1(0.1%) 0(0%) NS 0(0%) 1(0.2%) NS

POS 1248(99.9%) 114(100%) 587(100%) 522(99.8%)

CK18

NEG 54(4.7%) 4(4%) NS 10(1.9%) 35(7.2%) \0.001*

POS 1088(95.3%) 97(96%) 525(98.1%) 453(92.8%)

CK19

NEG 69(5.6%) 6(5.4%) NS 25(4.3%) 39(7.6%) 0.02

POS 1154(94.4%) 106(94.6%) 552(95.7%) 475(92.4%)

Basal cks

CK5

NEG 842(95.9%) 75(96.2%) NS 539(96.3%) 394(95.4%) NS

36(4.1%) 3(3.8%) 14(3.7%) 33(4.2%)POS

CK14

NEG 1078(92.9%) 104(92.9%) NS 503(93.8%) 463(92.6%) NS

POS 83(7.1%) 8(7.1%) 33(6.2%) 37(7.4%)

CK17

NEG 751(93.8%) 68(93.2%) NS 353(95.7%) 336(92.1%) 0.04

POS 50(6.2%) 5(6.8%) 16(4.3%) 29(7.9%)

Other ER-related proteins

FOXA1

NEG 338(44.2%) 42(45.7%) NS 147(42.6%) 171(48.9%) NS

POS 426(55.8%) 50(54.3%) 198(57.4%) 179(51.1%)

BEX1

NEG 226(30.1%) 29(33.3%) NS 95(29%) 108(32.5%0 NS

POS 524(69.9%) 58(66.7%) 233(71%) 224(67.5%)

AGTR1

NEG 157(24.2%) 8(11.4%) 0.04 72(25.6%) 56(19.6%) NS

LOW 205(31.6%) 23(32.9%) 89(31.7%) 86(30.1%)

HIGH 287(44.2%) 39(55.7%) 120(42.7%) 114(50.3%)

TFF1

NEG 432(63.8%) 45(54.9%) NS 191(64.3%) 188(61.2%) NS

245(36.2%) 37(45.1%) 106(35.7%) 119(38.8%)POS

TFF3

NEG 280(41.1%) 18(21.4%) \0.001* 118(40.7%) 115(36.2%) NS

POS 401(58.9%) 66(78.6%) 172(59.3%) 203(63.8%)

GATA3

NEG 320(48.7%) 41(59.4%) NS 143(49.3%) 160(50.8%) NS

POS 337(51.3%) 28(40.6%) 147(50.7%) 155(49.2%)

Breast Cancer Res Treat (2014) 145:317–330 321

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Table 4 continued

Markers HER2- HER2? p value Low Ki67 LI High Ki67 LI p value

ER- coregulators

CD71

NEG 411(52.6%) 25(30.5%) \0.001* 198(59.6%) 152(43.1%) \0.001*

POS 371(47.4%) 57(69.5%) 134(40.4%) 201(56.9%)

CARM1

NEG 237(31.4%) 19(20.9%) 0.05 111(33.1%) 92(27.3%) 0.01

LOW 388(51.4%) 49(53.8%) 176(52.5%) 168(49.9%)

HIGH 130(17.2%) 23(25.3%) 48(14.3%) 77(22.8%)

PELP1

NEG 157(19.8%) 7(8.5%) 0.04 79(24.1%) 63(17.2%) 0.02

LOW 517(65.1%) 61(74.4%) 212(64.6%) 244(66.5%)

HIGH 120(15.1%) 14(17.1%) 37(11.3%) 60(16.3%)

Cell cycle: Cyclin D1

NEG 191(58.2%) 9(34.6%) 0.01 76(55.1%) 59(51.3%) NS

POS 137(41.8%) 17(65.4%) 62(44.9%) 56(48.7%)

Tumor suppressor: p53

NEG 1039(83.1%) 67(59.3%) \0.001* 507(86.2%) 367(71%) \0.001*

POS 212(16.9%) 46(40.7%) 81(13.8%) 150(29%)

Proloferation markers

TK1

NEG 370(59.3%) 11(18.6%) \0.001* 189(70.5%) 124(40.5%) \0.001*

POS 254(40.7%) 48(81.4%) 79(29.5%) 182(59.5%)

Akt

NEG 167(21.1%) 22(26.5%) NS 78(21.8%) 73(20.6%) NS

POS 625(78.9%) 61(73.5%) 280(78.2%) 282(79.4%)

PI3K

NEG 284(30.5%) 19(20.2%) 0.005* 151(35.7%) 85(21.1%) \0.001*

LOW 280(30.1%) 22(23.4%) 3(2.6%) 109(27%)

HIGH 366(39.4%) 53(56.4%) 134(31.7%) 209(51.9%)

Apoptosis-related markers BCL2

NEG 91(13.2%) 25(35.2%) \0.001* 33(10.5%) 65(20.1%) 0.001*

POS 599(86.8%) 46(64.8%) 282(89.5%) 259(79.9%)

Cell adhesion molecules

E-Cadherin

NEG 461(37.8%) 28(25%) 0.00 215(37%) 169(33.5%) NS

757(62.2%) 84(75%) 7* 366(63%) 335(66.5%)POS

P-Cadherin

NEG 642(62.9%) 37(40.7%) \0.001* 321(67.7%) 255(57.3%) 0.001*

POS 378(37.1%) 54(59.3%) 153(32.3%) 190(42.7%)

N-Cadherin

NEG 461(37.8%) 28(25%) 0.007* 149(37.9%) 123(30.8%) 0.03

POS 757(62.2%) 84(75%) 244(62.1%) 276(69.2%)

Mucins: MUC-1

NEG 68(6.1%) 9(8.7%) NS 22(4.3%) 34(7%) 0.06

POS 1044(93.9%) 95(91.3%) 490(95.7%) 449(93%)

HER family proteins

322 Breast Cancer Res Treat (2014) 145:317–330

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Table 4 continued

Markers HER2- HER2? p value Low Ki67 LI High Ki67 LI p value

HER-1

NEG 1068(86.8%) 93(81.6%) NS 515(89.1%) 431(82.9%) 0.003*

POS 162(13.2%) 21(18.4%) 63(10.9%) 89(17.1%)

HER-3

NEG 113(10%) 11(10.7%) NS 62(11.9%) 28(5.8%) 0.001*

POS 1020(90%) 92(89.3%) 460(88.1%) 453(94.2%)

HER-4

NEG 206(16.9%) 13(11.8%) NS 115(20.4%) 55(10.6%) \0.001*

POS 1020(83.1%) 97(88.2%) 450(79.6%) 462(89.4%)

DNA repair genes

cH2AX

NEG 148(22.7%) 21(30%) NS 49(17.5%) 89(26.9%) 0.006*

POS 505(77.3%) 49(70%) 231(82.5%) 242(73.1%)

BARD1

NEG 681(92.2%) 73(81.1%) 0.001* 340(93%) 326(89.6%) NS

POS 58(7.8%) 17(18.9%) 23(7%) 38(10.4%)

RAD51

NEG 317(52.1%) 35(59.3%) NS 119(45.1%) 183(63.1%) \0.001*

POS 292(47.9%) 24(40.7%) 145(45.9%) 107(36.9%)

BRCA1

NEG 100(9.9%) 17(18.9%) 0.008* 44(9.3%) 64(14.4%) 0.016

POS 909(90.1%) 73(81.1%) 429(90.7%) 379(85.6%)

Table 5 Associations between high KI67-LI and clinicopathological variables within HER ? tumors

Clinicopathological variables HER? High KI67-LI

Low KI67LI High KI67-LI p value HER2- HER2? p value

Age

\50 5(25%) 37(49.3%) 0.05 154(35.2%) 37(49.3%) 0.01

[50 15(75%) 38(50.7%) 284(64.8%) 38(50.7%)

Menopausal status

Pre 5(25%) 39(52%) 0.03 163(37%) 39(52%) 0.01

Post 15(75%) 36(48%) 277(63%) 36(48%)

Size

\2 CM 10(50%) 31(41.3%) NS 205(46.8%) 31(41.3%) NS

[2 CM 10(50%) 44(58.7%) 233(53.2%) 44(58.7%)

Grade

1 3(15%) 0(0%) \0.001* 23(5.3%) 0(0%) \0.001*

2 8(40%) 11(14.7%) 157(35.8%) 11(14.7%)

3 9(45%) 64(85.3%) 258(58.9%) 64(85.3%)

Tubule

1 1(5.0%) 0(0.0%) 0.02 7(1.7%) 0(0.0%) 0.06

2 9(45.0%) 19(25.3%) 157(37.1%) 19(25.3%)

3 10(50.0%) 56(74.7%) 259(61.2%) 56(74.7%)

Pleomorphism

1 – – 0.002* 2(0.5%) 0(0.0%) 0.001*

2 8(42.1%) 9(12.0%) 137(32.4%) 9(12.0%)

3 11(57.9%) 66(88.0%) 284(67.1%) 66(88.0%)

Breast Cancer Res Treat (2014) 145:317–330 323

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blindly by the observer and according to the guidelines,

completing the HER2 PharmDXTM kit (Dako, UK).

Regarding KI67-LI was assessed by two pathologists

blinded to patients’ information and outcomes. The full-

face slide was scanned under low-power microscopy for

the area with the highest number of positive tumor nuclei

(hot spots), considering homogenous granular staining,

multiple-speckled staining nuclei, or noclular staining as

positive. Then, the LI was calculated (at high power

magnification 4009) as the percentage of positive tumor

cells from 1,000 malignant cells. Information regarding

scoring of different markers used in this study are as per-

formed as published before [30, 31]. This study was

approved by the Nottingham Research Ethics Committee.

Statistical analysis

SPSS version 21 (SPSS Inc., Chicago, IL, USA) was used

to analyze the data. For the parametric data, Chi-square test

was used to obtain the p value. Mann–Whitney test was

used to analyze the correlation between categorical and

Table 5 continued

Clinicopathological variables HER? High KI67-LI

Low KI67LI High KI67-LI p value HER2- HER2? p value

Mitosis

1 7(35.0%) 2(2.7%) \0.001* 66(15.6%) 2(2.7%) \0.001*

2 6(30.0%) 14(18.7 %) 125(29.6%) 14(18.7%)

3 7(35.0%) 59(78.7%) 232(54.8%) 59(78.7%)

Stage

1 15(75%) 34(45.3%) 0.04 240(54.9%) 34(45.3%) NS

2 3(15%) 33(44%) 157(35.9%) 33(44%)

3 2(10%) 8(10.7%) 40(9.2%) 8(10.7%)

Vascular invasion

NO 16(80%) 43(57.3%) 0.06 270(61.8%) 43(57.3%) NS

Definite 4(20%) 32(42.7%) 167(38.2%) 32(42.7%)

NPI

1 7(35.0%) 3(4.0%) \0.001* 91(20.8%) 3(4.0%) 0.001*

2 11(55.0%) 47(62.7%) 256(58.4%) 47(62.7%)

3 2(10.0%) 25(33.3%) 91(20.8%) 25(33.3%)

Table 6 Associations of high and low KI67-LI with different pro-

teins within HER2? cases

Markers HER2? HER2? p value

Low KI67-LI High KI67-LI

HER3

NEG 4(23.5%) 4(5.8%) 0.02

POS 1(76.5%) 65(94.2%)

TFF1

LOW 13(81.2%) 24(47.1%) 0.01

HIGH 3(18.8%) 27(52.9%)

FOXA1

NEG 12(70.6%) 26(41.9%) 0.03

POS 5(29.4%) 36(58.1%)

GATA3

NEG 10(83.3%) 22(51.2%) 0.04

POS 2(16.7%) 21(48.8%)

Table 7 Associations of HER2? and - with different proteins

within high KI67-LI

Markers High KI67 - High KI67-LI p value

HER2- HER2?

P53

NEG 321(74%) 40(54.8%) 0.001*

POS 113(26%) 33(45.2%)

TFF3

NEG 102(39.4%) 10(18.5%) 0.004*

POS 157(60.6%) 44(81.5%)

TFF1

NEG 159(63.9%) 24(47.1%) 0.02

POS 90(36.1%) 27(52.9%)

E-Cadherin

NEG 148(35.1%) 16(21.9%) 0.02

POS 274(64.9%) 57(78.1%)

324 Breast Cancer Res Treat (2014) 145:317–330

123

non-categorical data in which the distribution is not nor-

mal. Survival curves for BCSS and DMFS were drawn

using the Kaplan–Meier correlation and log-rank tests were

used to estimate the significance. Due to the large number

of parameters included in this study, a two-tailed p value

was considered significant if it was \0.01 and as a bor-

derline if p value was between 0.01 and 0.07.

Results

1,048 cases were informative for KI67 assessment and

1,401 were informative for HER2. 46.3 % of the total ER?

cases showed high KI67-LI and 8.4 % showed HER2

overexpression and both were positively correlated

(Table 2; p \ 0.001). Both KI67-LI and HER2 showed

positive associations with younger age, high-tumor grade

and high-NPI scores. High KI67-LI was associated with

larger size, advanced stage, and definite LVI (p \ 0.001)

while HER2? was associated with premenopausal status

(Table 3). Both high KI67-LI and HER2? were associated

with upregulation of ER-co-regulators (CD-71, CARM1,

and PELP1), proliferation-related markers (PI3KCA, TK1),

and the poor prognostic markers (P-cadherin and p53) and

with downregulation of androgen receptor (AR), Bcl2, and

ER levels.

Interestingly, high KI67-LI was associated with down-

regulation of luminal-enriched proteins (ck18, MUC1 core,

and FHIT proteins) and DNA-damage repair proteins

(cH2AX and RAD51; p \ 0.001) but with upregulation of

the other HER family proteins (HER1, HER3, and HER4).

On the other hand, HER2? was associated with upregu-

lation of the differentiation-associated proteins (TFF3,

E-cadherin and N-cadherin), BARD1 (p = 0.001), and

borderline upregulation of ER-related protein AGTR1,

(Table 4).

To further investigate the influence of HER2 on ER-

pathway, we repeated analysis on different subgroups.

Within high KI67-LI, HER2? was associated with high-

tumor grade and with upregulation of p53 and TFF3. A

trend for upregulation of TFF1 and E-cadherin was also

seen (Tables 5, 6, 7). High KI67-LI compared to HER2?/

low KI67-LI cases was negatively associated with luminal

and ER-related proteins: ck18 and TFF3 (p = 0.007 and

p = 0.002 respectively). In addition, it was associated

with a trend for upregulation of ck19, CD71, and

N-cadherin (p = 0.07, p = 0.03, and p = 0.02 respec-

tively; Table 8).

Outcome analyses

Univariate survival analyses revealed a strong association

between HER2? and high KI67-LI and poor outcome in

terms of shorter BCSS and DFI including shorter DMFS

(p \ 0.001; Fig. 1). Combined expression of HER2 and

KI67 showed that HER2? regardless of KI67-LI is asso-

ciated with the worst prognosis while low KI67-LI/HER2-

negative tumors showed the best outcome (p \ 0.001)

(Fig. 2). Multivariate analysis including tumor stage,

grade, size, and therapy showed that both HER2 and KI67-

LI maintained their independent prognostic significance.

When the analysis was restricted to subgroups, within

high KI67-LI, HER2 positivity was associated with poor

outcome in terms of BCSS at 15 years (Fig. 3). Within

HER2? subgroup, KI67-LI showed no significant associ-

ation with outcome. When the analysis was restricted to

show the effect of both proteins independently, HER2?/

low KI67-LI was associated with poor outcome in terms of

BCSS, DFI, and DMFS (Fig. 4).

Discussion

Molecular classification of BC and the concept of the ER?

luminal class and that ER, HER2, and proliferation are key

driving markers have attracted an attention in the clinical

community. However, the fact that luminal tumors com-

prise more than half of BC and HER2-positive tumors are

candidates for anti-HER2 therapy regardless of the

molecular class limits the clinical significance of this

classification. Although high proliferation is a feature of

Table 8 Associations between HER2?/low KI67-LI and high KI67-

LI/HER2- classes and different proteins

Markers HER2?/low KI67-LI High KI67-LI/HER2- p-value

CK18

NEG 29(5.6%) 160(25.6%) 0.007*

POS 34(94.4) 466(74.4%)

TFF3

NEG 7(25.9%) 219(56.9%) 0.002*

POS 20(74.1%) 166(43.1%)

Bcl2

NEG 12(60%) 130(32.9%) 0.01

POS 8(40%) 265(67.1%)

CD71

NEG 5(18.5%) 169(38.9%) 0.03

POS 22(81.5%) 266(61.1%)

N-Cadherin

NEG 2(8%) 147(29%) 0.02

POS 23(92%) 360(71%)

CK19

NEG 2(5.1%) 104(15.7%) 0.07

POS 37(94.9%) 557(84.3%)

Breast Cancer Res Treat (2014) 145:317–330 325

123

basal and HER2-positive tumors, a considerable number of

ER-positive tumors show high-proliferation status. To

identify the types of luminal tumors associated with poor

outcome, GEP studies have reported a luminal-B subclass

[32]. However, molecular features were used to define

these tumors varied among different studies and more than

one class have been identified [33–36]. In addition, the

availability and cost associated with GEP make identifi-

cation of a poor prognostic subclass of luminal tumors

difficult. Using surrogate immunohistochemical markers to

identify, a luminal-B class in routine clinical practice

appears a valid and practical alternative. However, previ-

ous studies have varied in the marker(s) used to identify

these tumors. Some studies have used the proliferation

marker KI67 [37, 38], HER2 [10, 11, 39–41], or both [13,

24, 37, 42] while others placed luminal HER2-positive

tumors in the HER2-positive subgroup [14]. Table 9,

illustrates different studies’ views regarding subclassifica-

tion of luminal groups.

Previous studies have indicated that the frequency of

ER-positive tumors with high proliferation status is greater

than that of HER2 positive tumors and that both markers

are associated with other poor prognostic features and

shorter survival. However, a comparative study of HER2

and KI67 in the luminal class regarding the clinical and

biological molecular features is lacking. In this study, we

confirm these findings and demonstrate that HER2 is

associated with a worse outcome independent of prolifer-

ation and other clinicopathological features. Importantly,

the aggressive behavior of HER2 positive tumors is not

associated with downregulation of luminal-enriched ER-

related biomarkers.

Fig. 1 Associations between

HER2, and KI67-LI and BCSS,

DFI and DMFS in months,

respectively

326 Breast Cancer Res Treat (2014) 145:317–330

123

Compared to tumors with high KI67-LI, HER2-positive

tumors retained their luminal-associated features as evi-

denced by positive association between HER2? with

TFF3, E-cadherin and borderline increase expression of

AGTR1. In contrast, high KI67-LI was associated with

downregulation of luminal markers (ck18, MUC1 core,

FHIT) and borderline significance with downregulation of

MUC1 protein and ck19 and borderline expression of basal

ck17. Although both HER2 and KI67 demonstrated asso-

ciations with features of poor prognosis, high KI67-LI and

not HER2? was significantly associated with clinical fea-

tures of advanced tumors including larger size, nodal

positivity, and definite LVI. In our study, high KI67-LI

showed an association with upregulation of other HER

family proteins (HER1, HER3, and HER4). Furthermore,

Within HER2? tumors, high KI67-LI maintained its

association with advanced stage and LVI which indicates

that high proliferation status is a feature of biological

aggressiveness rather than a unique driving genetic event.

Of note, although we and others [43] have noticed that

some HER2-positive tumors display prominent tubule/

gland formation despite high-nuclear grade, in this study,

both HER-positivity and high KI67-LI were associated

with loss of tubule formation and no significant difference

between them was identified.

Importantly, our results are in line with Staaf et al. [44]

who used a HER2 derived prognostic gene signature and

unsupervised analyses and identified three relatively equal

HER2-positive subgroups with different outcomes. One

subgroup not only tended to be more ER negative and was

associated with worse clinical outcome but it also had

Fig. 2 Associations between

HER2?ve/Low KI67-LI and

High KI67-LILHER2-ve and

BCSS, DFI, and DMFS,

respectively in months

Fig. 3 Association between HER2 within high KI67-LI and BCSS in

months

Breast Cancer Res Treat (2014) 145:317–330 327

123

predominant overexpression of steroid response genes

(steroid response–positive phenotype). The other two sub-

groups showed similar outcome but they differ in the

proliferation status. The subgroup that showed a high-

proliferation status with an active PI3 K signaling signa-

ture tended to be smaller in size with less nodal involve-

ment. Importantly, the predictor did not explicitly connect

to proliferation but recognizes a biologic phenomenon of

more general importance in BC progression including

genes associated with immune response, tumor invasion,

and metastasis.

With regard to outcome, although HER2 was not asso-

ciated with tumor size, lymph node stage, or downregulation

of luminal proteins, it was independently associated with

poorer outcome in terms of shorter BCSS, DFI, and DMFS

than high KI-67-LI (p = 0.001). This association is

observed in ER-positive tumors as well as in the ER-posi-

tive/high KI67-LI subgroup reflecting the association with

aggressive behavior independent on the proliferation status.

Importantly, within HER2-negative tumors, high KI67-LI

not only showed an association with downregulation of ER-

related/luminal-enriched molecular features and shorter

Fig. 4 Associations of

HER2?/Low KI67-LI and high

KI67-LI/HER2- and BCSS,

DFI, and DMFS, respectively in

months

Table 9 Definition of luminal-A and B by different studies

Studies Group 1 (luminal-A) Group 2 (luminal-B) Others

Cheang et al. [15] ER (?/-) and /or

PgR (?/)/ HER2-, low KI67

ER (?/-) and /or PgR (?/),

high KI67, HER2? in 30%

ER- HER2

Onitilo et al. [41] ER?/PgR?/HER2- ER?/PgR?/HER2?

Matos et al. [10] ER?/HER2?(0,?1,?2) ER?/HER2? (?3) –

Carey et al. [11] ER? and/or PgR?/HER2- ER?and/or PgR?/ HER2?(100%), p53?(23%)

Abd El-Rehim et al. [30] ER?/PgR?/ luminal CKS?/HER3,4,

low BRCA1, MUC-1?

ER?/PgR?/high BRCA1, MUC-1? –

Kurebayashi et al. [40] ER? and/or PgR?/HER2- ER? and/or PgR?/HER2? –

Ihemelandu et al. [39]

Hugh et al. [13] ER?/low HER2 and KI67 ER?/ high HER2 and KI67 –

328 Breast Cancer Res Treat (2014) 145:317–330

123

survival but also splits the cohort into two relatively equal

halves (43 and 57 % for high and low KI67-LI,

respectively).

In conclusion, the results of this study support the

hypothesis that HER2 gene amplification and protein over

expression occurs as a second oncogenic hit that drives the

molecular portrait and clinical behavior of ER-positive

HER2-positive BC independent of the ER-pathway or

proliferation. Our data indicate that HER2-positive ER-

positive BCs are distinct form of luminal-BC and that it

provides additional justification to place them in the HER2-

positive class candidate for anti-HER2 therapy. ER-posi-

tive/HER2-negative tumors can be stratified into two rel-

atively equal subgroups with distinct outcome using KI67-

LI.

Acknowledgements Dena A Jerjees is funded by the higher com-

mittee of educational development in Iraq.

Conflicts of interest None.

Ethical standards This study was approved by the Nottingham

Research Ethics Committee.

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