ORIGINAL ARTICLE / ARTICLE ORIGINAL

Helix-loop-helix protein inhibitor of differentiation 4 (ID4) expression isan indicator of poor survival in Nigerian breast cancer women

L’expression de l’inhibiteur de différenciation 4 (ID4) lié à une protéine hélice-boucle-héliceconstitue un indicateur d’un taux de survie faible chez les femmes nigérianes atteintesd’un cancer du sein

A.O.J. Agboola · A.A.F. Banjo · C. Anunobi · B. Salami · M. Deji-Agboola · A. Musa · C.C. Nolan · E.A. Rakha ·I.O. Ellis · A.R. Green

Received: 17 June 2013; Accepted: 9 September 2013© Springer-Verlag France 2013

Abstract Aim: This study investigates the prognostic featuresof helix-loop-helix protein inhibitor of differentiation 4 (ID4), anegative regulator of breast cancer-associated gene 1 (BRCA1)in breast cancer from Nigerian women, where the mode oftreatments remains unclear.Materials and methods: In this study, the protein expressionof ID4 was investigated in a series of 235 Nigerian women,prepared as a tissue microarray (TMA), using immunohis-tochemistry.Results: There was a positive correlation between ID4 expres-sion and variables associated with poor prognosis and basal-phenotype including high histological tumour grade, expres-sion of epidermal growth factor receptor (EGFR), cytokera-tins (CK5/6, CK14), placental cadherin (P-cadherin), p53 andPhosphoinositide-3-kinases (PI3KCA). A greater proportionof ID4 positive tumours lacked expression of BRCA1, oestro-gen receptor (ER) and progesterone receptor (PR). Univariate

and multivariate survival analysis showed that ID4 remaineda prognostic factor for breast cancer-specific survival (BCSS)independent of tumour grade, size, lymph node involvementand basal-like phenotype.Conclusion: ID4 may play a role in the development andprogression of breast cancer in Nigerian women. ID4 couldalso potentially be used as a target for drug therapy in thispoor prognostic ethnic group.

Keywords ID4 · Independent prognostic factor · Breastcancer · Nigerian · Basal-like · Ethnicity

Résumé Objectif : Cette étude examine le pronostic del’inhibiteur de différenciation 4 (ID4) d’une protéine hélice-boucle-hélice, un régulateur négatif du gène 1 associé au can-cer du sein (BRCA1) chez des femmes nigérianes atteintesd’un cancer du sein et chez lesquelles le mode de traitementdemeure incertain.Matériel et méthodes : Dans cette étude, l’expression desprotéines de l’ID4 a été examinée sur 235 femmes nigér-ianes et préparée en matrices tissulaires (TMA), à l’aide del’immunohistochimie.Résultats : Une corrélation positive a été obtenue entrel’expression de l’ID4 et les variables liées à un pronosticdéfavorable et au phénotype de type basaloïde, y comprisun type histologique élevé de tumeurs, l’expression durécepteur de facteur de croissance épidermique (EGFR),les cytokératines (CK5/6, CK14), la cadhérine dans leplacenta (P-cadhérine), la voie p53 et les phosphoinositides3-kinases (PI3KCA). Une proportion plus importante detumeurs positives à l’ID4 n’exprimaient pas le gène 1 ducancer du sein (BRCA1), le récepteur des estrogènes (ER)et le récepteur de la progestérone (PR). Les analyses de sur-vie univariées et multivariées ont montré que l’ID4 restaitun facteur pronostique des chances de survie au cancer du

A.O.J. Agboola (*) · C.C. Nolan · E.A. Rakha · I.O. Ellis ·A.R. GreenDivision of Pathology, School of Molecular Medical Sciences,Nottingham University Hospitals and University of Nottingham,Nottingham, United Kingdome-mail : johndeji2001@yahoo.co.uk

B. Salami · A. MusaDept of Surgery, Olabisi Onabanjo University and OlabisiOnabanjo University Teaching Hospital, Sagamu, Nigeria

A.O.J. Agboola · A.A.F. Banjo · C. AnunobiDept of Morbid Anatomy and Histopathology,Olabisi Onabanjo University and Olabisi Onabanjo UniversityTeaching Hospital,Sagamu, Nigeria

M. Deji-AgboolaDept of Medical Microbiology parasitology,Olabisi Onabanjo University, Sagamu, Nigeria

J. Afr. CancerDOI 10.1007/s12558-013-0298-1

sein, indépendamment du type, de la taille, de l’infiltra-tion ganglionnaire et du phénotype de type basaloïde de latumeur.Conclusion : Il se peut que l’ID4 joue un rôle dans le dével-oppement et la progression du cancer du sein chez lesfemmes nigérianes. L’ID4 pourrait également être utilisé entant qu’objectif pour la pharmacothérapie dans ce groupeethnique au pronostic défavorable.

Mots clés ID4 · Facteur pronostique indépendant · Cancerdu sein · Nigériane · Basaloïde · Ethnicité

Introduction

Breast cancer-associated gene 1 (BRCA1) has a major role inDNA repair where it binds to DNA branch structures andinduces chromatin unfolding, thereby participating in thetranscription and repair [1]. Low BRCA1 expression in spo-radic tumours has been linked with high tumour grade,higher proliferation rate and risk of metastases resulting inpoor clinical outcome including early recurrence and shortersurvival [2,3]. BRCA1 dysfunction in sporadic tumours hasbeen related to its regulation by either associated proteins orepigenetic silencing [4,5]. Several BRCA1 regulatory pro-teins have been identified in breast cancer and they arealso found to be associated with cellular changes in tumour-igenesis [2,6].

Helix-loop-helix protein inhibitor of differentiation 4(ID4) is a regulator of BRCA1 [4,7]. The ID4 gene is locatedon 6p22.3 [8] and its protein product has diverse functionsincluding cellular proliferation and differentiation [9,10].ID4 acts as a negative regulator of transcription uponhetero-dimerisation with other transcription proteins andalso has the capability of binding to non-helix-loop-helixtranscription proteins including the paired box (PAX) pro-teins and retinoblastoma, both involved with cellular prolif-eration and differentiation [9,10]. The roles of ID4 appear tobe organ specific, for instance, during embryogenesis, itsdistribution is only found in the nervous system where itsexpression is necessary for normal development [11]. Simi-larly, in testicular cells, it increases in response to differenti-ating agents [12], while in astrocytes, a decrease in itsexpression is required [13].

In cancer, ID4 mRNA expression is increased in breastcarcinoma compared with benign and normal tissue and itis associated with increased proliferation and invasivenessof the tumour cells [14].

ID4 is known to be a regulator of BRCA1 and oestrogenreceptor (ER) in breast carcinomas but its exact roleremains speculative. The relationship between ID4 andBRCA1 was first established by Berger et al [4], where

an over expression of ID4 in target cells caused down-regulation of BRCA1 in human MCF-7 breast cancercells treated with 17β-oestradiol. In their study, ID4 RNAexpression was reduced more than two-folds followingoestrogen stimulation and the ratio of BRCA1 mRNA toID4 mRNA increased seven-fold by this stimulation [4]. Asimilar finding was reported by Roldan et al. [15]. ID4 hastherefore been hypothesised as a potential tumour suppres-sor gene that regulates oestrogen expression in breast duc-tal epithelium [4,15]. However, in contrast to above find-ings, Welsh et al reported direct correlation betweenincreased BRCA1 and ID4 expression [16]. Previous stud-ies of the prognostic significance of ID4 in cancer demon-strated that it is associated with clinicopathological para-meters predictor of unfavourable prognosis in breast [17]and colorectal cancers [18].

Black women with breast cancer have a higher risk forearly onset, with a high mortality rate, prompting specula-tion that risk factors could be genetic and the molecularportrait of these tumours are likely to be different tothose of Caucasian women [19]. Advances in the under-standing of molecular basis of breast cancer among differ-ent racial backgrounds show that Black women are associ-ated with triple negative breast cancer (TNBC), basal-likephenotype and BRCA1 deficiency [19]. The exact role ofBRCA1dysfunction in Black women that harbour a greaterpercentage of BRCA1 tumours is yet to be fully under-stood. Consequently, the aim of this study is to biologi-cally characterise the ID4 protein expression in relationwith clinicopathological parameters and clinical outcomein an indigenous population of Nigerian women withbreast cancer (BC) in order to contribute better towardsour understanding of the mechanisms of BC developmentin ethnic women.

Materials and methods

Patients

The patient cohort comprised formalin-fixed paraffin-embedded (FFPE) breast cases from 235 women present-ing at the Olabisi Onabanjo University Teaching Hospital,Sagamu, and Histopathology Specialist Laboratory, Idi-Araba Lagos, Nigeria, from January 2002 to December2008. Clinical history and tumour characteristics includingage, menopausal status, tumour type, histological grade,tumour size, lymph node status and vascular invasionwere assessed in a standardised manner for all patients.

Patient outcome and treatment data were retrieved fromthe patient’s records. All patients were treated with a combi-nation of classical chemotherapy (cyclophosphamide, meth-otrexate and 5FU) and hormonal therapy (tamoxifen).

2 J. Afr. Cancer

Eighty five of the patients (42.5%) received radiotherapy.Patients were followed up for at least 60 months.

The Reporting Recommendations for Tumour MarkerPrognostic Studies (REMARK) criteria, recommended byMcShane et al. [20], were followed. This study was approvedby the Medical Advisory Committee, Olabisi Onabanjo Uni-versity Teaching Hospital, and by the Nottingham ResearchEthics Committee 2 under the title of “Development of amolecular genetics classification of breast cancer”.

Tissue microarray array construction

Two hundred and thirty-five samples from the Nigeriancohort were constructed as tissue microarrays (TMAs) aspreviously described [19]. Breast tumour cores were takenfrom each FFPE donor tissue block that has been marked forthe most representative points of tumour (both peripherallyand centrally). A precision instrument (ALPHELYS Mini-Core®) was used to take representative cores of tissue(0.6 mm diameter, 3 mm height) from each sample, whichwas arrayed into a recipient paraffin.

Immunohistochemistry method

Table 1 shows the different biomarkers included in thisstudy. All the biomarkers required antigen retrieval exceptc-erbB2, EGFR and laminin. Antigen retrieval was per-formed by microwaving the slides at 800 W for 10 min fol-lowed by 560 W for 10 min in citrate buffer (1M sodiumcitrate at a pH of 6.0) followed by cooling in running waterimmediately. The primary antibody for the biomarkers wasincubated for 60 min at room temperature. Diaminobenzi-dine tetrahydrochloride (DAB) solution was incubated for10 min after which copper-sulphate solution (0.5% coppersulphate in 0.8% sodium chloride) was applied to the slidesand incubated for 10 min each and counterstained with hae-matoxylin for 2–3 min, followed by rinsing in tap water.Slides were de-hydrated by immersing in three alcoholbaths for 10 s and cleared in two xylene baths followed byapplication of cover slip. Negative and positive controlswere performed by omitting the primary antibody andincluding control tissues as specified by the antibody sup-plier, respectively (Table1).

Immunohistochemical scoring

The scoring was performed using the percentage of posi-tive cells to produce a final score in the range 0–100. Thecases were scored without knowledge of the clinicopatho-logical parameters or patient outcome. TMAs were scoredindependently twice by one observer (JA). The mean of thescores were calculated to reach a final score. A proportionof these were counterscored by an observer (AG) to ensure

reproducibility. The biomarkers were dichotomised intogroups according to the median of percentage staining:0–49% as negative/low expression and above 49% as posi-tive expression.

For c-erbB2 (HER2), the American Society of ClinicalOncology/College of American Pathologists GuidelineRecommendations for HER2 Testing in Breast Cancer wasused for assessment [21]. Equivocal (2+) cases were con-firmed by chromogenic in-situ hybridization (CISH) as pre-viously described [22]. For molecular classification, Niel-sen’s method [23] was used. This comprises Luminal A(ER, PR positive and HER2 negative), Luminal B (ER, PRHER2 positive), Basal (ER, PR, HER2 negative and CK5/6and/or EGFR positive), HER2 (ER negative and HER2 pos-itive) and an unclassified group (ER, PR, HER2 CK5/6 andEGFR negative).

Statistical analysis

Statistical analysis was performed using SPSS 16.0 statis-tical software. Chi-squared analyses were used for inter-relationships between the ID4 expression, clinicopatholog-ical parameters and other biomarkers. The Kaplan–Meiersurvival method and the log-rank test were used for sur-vival curves. Multivariate analyses using Cox proportionalhazard regression models were performed and from themodel both the risk factor and 95% confidence intervalswere generated. A two-sided p-value of <0.05 was consid-ered significant.

Results

TMA microscopical examination revealed a variety ofnuclear staining localities. ID4 immunoreactivity wasobserved in the nucleus of breast tumour cells with differentintensities and proportion of cells stained (Fig. 1). With thesecut off points of percentage staining of 0–49% as negative/low and above 49% as positive expression, out of 235 cases,156 (66.4%) and 79 (33.6%) were considered positive andnegative/low, respectively (Table 2). The relationshipbetween expression of ID4 and clinicopathological variablesis summarised in Table 3. There was a significant positivecorrelation between ID4 expression and high histologicaltumour grade (p = 0.003) and mitotic frequency (p =0.002). There was no significant association with meno-pausal status, age at diagnosis, tumour histological type,size, lymph node status or presence of lymphovascularinvasion.

The relationship between ID4 and other biomarkers issummarised in Table 4. A significantly greater proportionof ID4 positive tumours lacked expression of BRCA1(p < 0.001). ID4 expression was also inversely correlated

J. Afr. Cancer 3

Tab

le1

Sources,d

ilutio

n,distributio

n,cut-offpointsandpre-treatm

entused

forrevalid

ation.

Antibod

yClone

Sou

rce

Dilution

Distribution

ScoringSystem

Cut-offs

Pre-treatmen

tPositivecontrol

BRCA1

Ab-1(M

S110)

Calbiochem

1:15

0Nuclear

%of

positive

cells

<25

%(negative)

Antigen

retrieval

microwave

MCF7cells(hum

an

breast

adenocarcino

ma

cellline)

Ck5

/6M72

37Dako-

Cytom

ation

1:60

Cytop

lasm

%of

positive

cells

≥10

%(positive)

Antigen

retrieval

microwave

Kno

wncase

ofCK56

BC

Ck1

4LL00

2Nov

ocastra

1:40

Cytop

lasm

%of

positive

cells

≥10

%(positive)

Antigen

retrieval

microwave

Kno

wncase

ofCK14

breast

cancer

E-cadherin

NCH-38

Dako-

Cytom

ation

1:10

0Cytop

lasm

andmem

brane

%of

positive

cells

≥10

0H

score

(positive)

Antigen

retrieval

microwave

Normal

gastricmucosa

EGFR

31G7

Nov

ocastra

1:30

Mem

brane

%of

positive

cells

≥10

%(positive)

Not

requ

ired

Myo

epithelial

cells

ofno

rmaldu

ctin

norm

al

mam

marygland

erbB

2Polyclonal

Dako-

Cytom

ation

1:10

0Mem

brane

Table

2.1

Table

2.1

Not

requ

ired

Kno

wncase

oferbB

2

strong

BC

expression

ER

1D5

Dako-

Cytom

ation

1:20

0Nuclear

%of

positive

cells

≥0(po

sitive)

Antigen

retrieval

microwave

Normal

breast

acini

ID4

Ab7

7345

Abcam

1:10

0Nuclear

%of

positive

cells

≥50

%(positive)

Antigen

retrieval

microwave

Hum

ancoloncancer

Lam

inin

Ab4

9726

Abcam

1:50

Cytop

lasm

andmem

brane

%of

positive

cells

≥1%

(positive)

Antigen

retrieval

ProteinaseK

Kidneytissue

MTA1

Ab8

4136

Abcam

1:10

0Nuclear

%of

positive

cells

≥1%

(positive)

Antigen

retrieval

microwave

Hum

angastric

adenocarcino

ma

P-cadherin

NCL-

P-cad

Nov

ocastra

1:20

0Cytop

lasm

%of

positive

cells

≥5%

(positive)

Antigen

retrieval

microwave

Kno

wncase

ofP-

cadh

erin

strong

BC

expression

PR

PR

Dako-

Cytom

ation

1:15

0Nuclear

%of

positive

cells

≥0(positive)

Antigen

retrieval

microwave

Normal

breast

acini

P13

KCA

HPA

0009

985

Sigma

1:50

Nuclear

%of

positive

cells

>60

%(positive)

Antigen

retrieval

microwave

Hum

anbreastcarcinom

a

p53

DO7

Nov

ocastra

1:50

Nuclear

%of

positive

cells

>10

%(negative)

Antigen

retrieval

microwave

Normal

breast

acini

4 J. Afr. Cancer

with steroid hormone receptors, with majority of ID4 posi-tive tumours either showing absence or low levels of ER orprogesterone receptor (PR) (all p < 0.001). When comparedwith HER family, although epidermal growth factor receptor(EGFR) was considerably expressed in ID4 positive

tumours (p = 0.001), there was no correlation betweenHER-2 and ID4. Furthermore, the majority of the tumoursthat expressed ID4 were positively correlated with theexpression of basal cytokeratin expressions (CK5/6 (p <0.001) and CK14 (p = 0.004)), p53 (p < 0.001), placental-cadherin (P-cadherin) (p = 0.004) and phosphoinositide-3-kinases (PI3KCA (p < 0.001)). Regarding BC molecularclasses, ID4 expression was significantly associated withtriple negative and basal-like phenotype compared with theluminal or HER-2 phenotypes (p < 0.001). Univariate sur-vival analysis showed that tumours positive for ID4 had asignificantly shorter BCSS (p < 0.001) and disease-freeinterval (DFI) (p = 0.03) (Fig. 2). In Cox multivariate analy-sis, ID4 remained a prognostic factor for BCSS (p = 0.001)independent of tumour grade, size, lymph node involvementand basal-like phenotype (Table 5).

Discussion

There are concerted efforts to understand mechanisms asso-ciated with BRCA1 dysfunction particularly in basal-likeBCs, which is more commonly observed in the Black nation-alities [19].

In this study, expression of ID4 was analysed in relationto clinicopathological parameters, clinical outcome and bio-marker expression in BCs from Nigerian women to charac-terise and determine its prognostic significance. In agree-ment with previous findings, ID4 protein was expressedin the nucleus [4,7,16]. ID4 expression was associatedwith tumours of aggressive features and these tumourslack steroid hormones receptor proteins, which are consis-tent with the previous findings [4,7,24]. In addition, thisbiomarker was highly expressed in basal-like BCs, asdefined by Nielsen’s classification and TNBCs; this isalso in agreement with a study, where ID4 was reportedto be strongly associated with TNBC and basal cytokeratin(CK14) [7,25].

ID4 was also associated with loss of or reduced BRCA1expression in agreement with previous studies [4,15].BRCA1 is essential for preserving the genomic integrity,cell cycle checkpoints and DNA repairs [1]. Alterednuclear expression of BRCA1 causes a defect in the main-tenance of normal genomic integrity and therefore predis-posing the cells into abnormal chromatin modification,defective cell cycle checkpoints, aneuploidy and highrisk of aggressive BC [1]. Altered nuclear expression ofBRCA1 has several lines of evidence to suggest that it islinked with basal and TNBC, where the majority of theTNBCs express basal CKs, and also lack BRCA1 [26].This association between the BRCA1-related, basal-likephenotype and TNBC suggests that hormonal factorsplay a critical role in the development of this type of BC

Table 2 Frequency of ID4 expression in Nigerian breast

cancer.

Biomarkers Frequency %

Negative 79 33.6

Positive 156 66.4

Total 235 100

Fig. 1 ID4 (A) positive and (B) negative immunoreactivity

in Nigerian breast cancer. Magnification ×20

J. Afr. Cancer 5

and by extension their response to hormonal treatment[26]. ID4 may therefore be involved in the disruption ofBRCA1 molecular pathways which might contribute tothe aggressive form of BC observed in Black women. Fur-

thermore, ID4 positive tumours would probably notrespond well to hormonal therapy being associated withbasal-like and triple negative tumours. Therefore, ID4expression could influence the poor response of TNBC

Table 3 Relationship between ID4 expression and clinicopathological variables in Nigeria breast cancer.

Variables ID4 expression

Negative/Low (%) Positive (%) χ2 value p-value

Age (years)

≤50 53 (67.1) 105 (67.3) 0.01 0.97

>50 26 (32.9) 51 (32.7)

Lymph node involvement

Negative 8 (10.1) 10 (6.4) 1.03 0.31

Positive 71 (89.9) 146 (93.6)

Menopausal

Pre 51 (64.6) 114 (73.1) 1.82 0.17

Post 28 (35.4) 42 (26.9)

Mitotic frequency

Low 62 (78.5) 85 (54.5) 12.89 0.002

Medium 10 (12.7) 42 (26.9)

High 7 (8.8) 29 (18.6)

Nuclear pleomorphism

Small uniform cells 0 (0.0) 1 (0.6) 1.14 0.56

Moderate increase in size 28 (35.4) 47 (30.2)

Marked variation 51 (64.6) 108 (69.2)

Tubule formation

>75% 2 (2.5) 1 (0.6) 1.54 0.46

10–75% 4 (5.1) 9 (5.8)

<10% 73 (92.4) 146 (93.6)

Tumour grade

1 3 (3.8) 4 (2.6) 11.66 0.003

2 59 (74.7) 83 (53.2)

3 17 (21.5) 69 (44.2)

Tumour type

Invasive ductal 64 (81.0) 140 (89.8) 9.89 0.27

Tubular mixed 7 (8.8) 6 (3.8)

Lobular mixed 2 (2.5) 2 (1.3)

Classical lobular 1 (1.3) 2 (1.3)

Mucinous 2 (2.5) 1 (0.6)

Atypical medullary 1 (1.3) 3 (1.9)

Tubular 0 (0.0) 2 (1.3)

Typical medullary 1 (1.3) 0 (0.0)

Tubulolobular 1 (1.3) 0 (0.0)

Tumour size (cm)

≤2 4 (5.1) 16 (10.3) 1.81 0.17

>2 75 (94.9) 140 (89.7)

Vascular invasion

Negative 55 (69.6) 117 (75.0) 0.77 0.37

Positive 24 (30.4) 39 (25.0)

Bold figures represent the statistical significant values.

6 J. Afr. Cancer

in Black women to both chemotherapy and hormonal ther-apy and subsequently contribute to the poor patient out-come observed in this study.

ID4 was also positively associated with p53 expression.The p53 gene has a negative feedback mechanism on itsdownstream effectors influencing cells with many physio-logical and pathological consequences [27]. An increase inp53 expression encourages the accumulation of p53 that pre-disposes the cell to aggressive forms of tumour with pooroutcome [27]. Data suggests that tumours expressing ID4might encourage the accumulation of abnormal p53 in the

Table 4 Relationship between ID4 and other biomarkers

in Nigerian breast cancer.

ID4 expression

Variables Negative (%) Positive (%) χ2 value p-value

BRCA1

Negative 42 (70.0) 119 (90.8) 13.49 0.001

Positive 18 (30.0) 12 (9.2)

Basal cytokeratins

Ck5/6

Negative 54 (84.5) 56 (40.6) 33.81 <0.001

Positive 10 (15.5) 82 (59.4)

CK14

Negative 39 (70.9) 61 (48.0) 8.14 0.004

Positive 16 (29.1) 66 (52.0)

Cell adhesion molecules

E-cadherin

Negative 37 (68.5) 82 (67.8) 0.01 0.92

Positive 17 (31.5) 39 (32.2)

P-cadherin

Negative 32 (56.1) 40 (33.6) 8.09 0.004

Positive 25 (43.9) 79 (66.4)

Cell cycle markers

P53

Negative 26 (51.0) 23 (17.4) 21.13 <0.001

Positive 25 (49.0) 109 (82.6)

HER family

EGFR

Negative 45 (80.4) 75 (54.7) 11.08 0.001

Positive 11 (19.6) 62 (45.3)

HER-2

Negative 47 (75.8) 115 (82.7) 1.31 0.25

Positive 15 (24.2) 24 (17.3)

P13KCA

Negative 43 (60.6) 49 (33.8) 13.97 <0.001

Positive 28 (39.4) 96 (66.2)

Steroid hormone receptors

ER

Negative 39 (56.5) 130 (86.7) 24.38 <0.001

Positive 30 (43.5) 20 (13.3)

PR

Negative 28 (52.8) 117 (90.0) 31.62 <0.001

Positive 25 (47.2) 13 (10.0)

Molecular classification

Basal 3 (8.4) 67 (65.7) 52.70 <0.001

HER2 7 (19.4) 19 (18.6)

Luminal A 19 (52.8) 14 (13.7)

Luminal B 7 (19.4) 2 (2.0)

Triple negative

Negative 40 (47.6) 12 (12.2) 27.73 <0.001

Positive 44 (52.4) 86 (87.8)

Bold figures represent the statistical significant values.

Fig. 2 ID4 expression in relation to (A) breast cancer-specific sur-

vival (BCSS) (B) disease-free interval (DFI) demonstrating out-

come differences in Nigerian BC

Bold figures represent the statistical significant values

J. Afr. Cancer 7

cell, and thus, ID4 with the co-expression of p53 could con-tribute to the aggressiveness of the tumour malfunctioning ofthe apoptotic pathway [28].

ID4 positive expression was also associated with theexpression of P-cadherin and PI3KCA and this is consistentwith the previous study on epithelial to mesenchymal transi-tion (EMT) expression in BC [28]. Loss of E-cadherin andpositive P-cadherin or N-cadherin expression is an impor-tant step in the tumour metastasis [29]. A previous studyshowed that over expression of N-cadherin, P-cadherin andlaminin in conjunction with reduction or loss of E-cadherinare more likely to occur in basal-like BC [29]. In addition,PI3KCA acts as transcriptional repressor of E-cadherin [30],thereby switching tumour cells from the cohesive nature tohighly motile cells necessary for cancer cell haematogenousand lymphatic metastasis. Therefore, ID4 might probablycontribute to the high levels of metastatic spread observedamong Black women with BC.

In conclusion, the results in this study confirm that ID4 isa potential down regulator of BRCA1 particularly in Blackwomen where loss of BRCA1 expression was observed ina larger proportion of cases. In addition, this marker couldbe used to screen for BRCA1 dysfunction and basal-liketumours.

Acknowledgement I wish to acknowledge the efforts of thestaffs of the Histopathology department, Olabisi OnabanjoUniversity Teaching Hospital, Sagamu, Nigeria, and Univer-sity of Nottingham City Hospital for the completion of thisproject. My appreciation also goes to my colleagues for theirteam spirit and the University of Nottingham, where thisstudy was carried out.

Authors’ contributions

AJA participated in the design of the study, performed theimmunohistochemistry and manuscript write up; AAM and

BSS contributed to patients’ management and outcomefollow up; AAB performed the histological diagnosis ofthe samples in Nigeria; ERK and IOE contributed to there-evaluation of the histological diagnosis of the samplesfrom Nigeria at UK histopathology laboratory; and IOEalso contributed immensely towards the design of thestudy and manuscript development. CCN and AG per-formed the TMA, and additionally, AG participated inthe design of the study, immunohistochemical scoringand also edited the manuscript. AMD performed the statis-tical analysis.

Conflict of interests: A.O.J. Agboola, A.A.F. Banjo,C. Anunobi, B. Salami, M. Deji-Agboola, A. Musa,C.C. Nolan, E.A. Rakha, I.O. Ellis and A.R. Green haveno conflict of interest to declare.

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2. Seery LT, Knowlden JM, Gee JM, et al (1999) BRCA1 expres-sion levels predict distant metastasis of sporadic breast cancers.Int J Cancer 84(3):258–62

3. Lee WY (2002) Frequent loss of BRCA1 nuclear expression inyoung women with breast cancer: an immunohistochemical studyfrom an area of low incidence but early onset. Appl Immunohis-tochem Mol Morphol 10(4):310–5

4. Beger C, Pierce LN, Kruger M, et al (2001) Identification ofId4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach. Proc Natl Acad SciU S A 98(1):130–5

5. Garcia AI, Buisson M, Bertrand P, et al (2011) Down-regulationof BRCA1 expression by miR-146a and miR-146b-5p in triplenegative sporadic breast cancers. EMBO Mol Med 3(5):279–90

6. Jarvis EM, Kirk JA, Clarke CL et al (1998) Loss of nuclearBRCA1 expression in breast cancers is associated with a highlyproliferative tumor phenotype. Cancer Genet Cytogenet 101(2):109–15

Table 5 Cox multivariate analysis of probability of breast cancer-specific survival (BCSS) and disease-free interval (DFI) in Nigerian

breast cancer series with ID4 expression.

BCSS DFI

Variables p-value Hazard ratio 95% CI p-value Hazard ratio 95% CI

Lower Upper Lower Upper

ID4 0.001 2.42 1.46 4.00 0.52 1.25 0.62 2.54

Basal-like phenotype 0.69 1.09 0.69 1.73 0.71 0.88 0.46 1.70

Grade <0.001 2.06 1.38 3.07 <0.001 4.97 2.59 9.49

Lymph node 0.42 1.42 0.60 3.34 0.10 5.40 0.70 41.64

Tumour size 0.75 0.88 0.42 1.85 0.17 0.52 0.20 1.34

Bold figures represent the statistical significant values.

8 J. Afr. Cancer

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