COMPARISON OF OESTROGEN RECEPTOR, PROGESTERONE RECEPTOR, AND HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR-2 EXPRESSION IN PRIMARY BREAST CANCER AND LYMPH NODE METASTASIS AT THE KORLE-BU TEACHING HOSPITAL, ACCRA, GHANA. BERNICE ANANE MAWULI ID NO.: 10357510 THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL. IMMUNOLOGY DEGREE. JULY, 2013 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh I DECLARATION BY THE CANDIDATE I her eb y de clar e that thi s is the product of m y own rese arch undert aken und er supe rvision and has neit her been pr e sented in whole nor in part for another de gre e el sewher e. I am responsi ble for an y flaws in this work. Signa t ure…………………… Date………. /……../……….. (Bernic e Anane Mawul i ) DECLARATION BY THE SUPERVISORS We here by dec l a re tha t the prac ti ca l work and pre se nt at i on of this the si s were supe rvi se d in acc orda nc e with guid e li nes on supe rvi si on of the si s laid down by the Unive rsi t y of Gha na . Princ i pa l supe rvi sor Signature…………………… Date……. /……../……….. (Professor Yao Tett e y) Co -supervisor Signature…………………… Date……. /……../……….. (P rofessor And rew Anth on y Adjei ) University of Ghana http://ugspace.ug.edu.gh II DEDICATION I dedicate this work to th e foll owing indi viduals; M y parents, Mr. Fran cis Anane Ma wuli and Mada m Merc y Koduah. M y sibl ings, Emelia Ank amah, Wil li am A. Mawuli , King Gideon Safo and King Kwadwo Safo. All women wit h breast cancer and their various fa mi li es. University of Ghana http://ugspace.ug.edu.gh III ACKNOWLEDGEMENTS M y sinc er e app reci ati on go es to Apostl e Dr. Kwadwo Sa fo fo r his pro f ound assi stance throughout m y academi c lif e. I am most grate ful to Pro fessor Andrew Anthon y Adjei for all the en coura gem ent he gav e me throughout m y MPhil . Progr am me and also fo r the co super visi on of my wo rk. To Professor Richard K. Gya si, the Head o f the Department of Pathol o g y, I appr eciat e al l the encoura gement and the assi stance giv en me to ensure the successful compl eti on of m y work. I am most grateful to Pr ofessor Yao Tett e y fo r t he wonde rful sup ervision of m y work to compl eti on. I sa y a big thank you to Dr. Lawren ce Edusei of the Departmen t of Pathol og y for reviewin g m y immunos tained sli des, and to Dr. Edmund Der, also of t he Pathol og y Department fo r assi sti ng in the selecti on of m y cas es. I am grat eful to Profess or Clegg - Lampt e y of the Surgi cal dep artment of The Korle -B u Teachin g Hospit al for assi sti ng in the provisi on of information on the mana gement of pati ents with breast can c er at the Hospit al. To Dr. Alf red Ed win Yawson of the Comm unity Healt h Department of The Korl e - Bu Teachin g Hospit al, I sa y a big thank you fo r assi sti ng in the stat isti cal anal yses of thi s thesis. To Raz ak of the Procur ement Unit of the Universit y of Ghana M edical School, I am gr ateful fo r the prompt pr ocessi ng of the pro curem ent documents for m y rea gents. To Mr. Berko and Afi of Huge Lim it ed, I sa y th anks for supp l yin g the rea gents for m y work. University of Ghana http://ugspace.ug.edu.gh IV To Ama Afr ah and Cecil ia Smi th of the Pathol ogy Depa rtment, I appr ecia te all the piec es of advice conce rnin g m y work. I am grat eful to Richard Hooper, also of the Patholog y Depa rtment, for assi sti ng in the bench work. M y warmest appr eciation goes to Th e Scholarship Comm it tee of the Coll ege of Healt h Sciences , Korl e- Bu for th e awa rd of a rese arch gra nt to support part of the funding of this work. Finall y, man y thanks go to Mrs. Emelia Abbe yqu a ye of the Depa rtment of Pathol ogy fo r assi sti ng in the secr etari a l aspect of this work. University of Ghana http://ugspace.ug.edu.gh V TABLE OF CONTENTS DECLARATION…………………………………………………………………………..I DEDIC AT I ON…………………………………………………………………………....II ACKNOW LED GE MENTS ……………………………………………………………..III TABLE OF CO NTENTS…………………………………………………………………V LIST OF TABLES…………………………………………………………………….… IX LIST OF FIGURES………………………………………………………………………X LIST OF ABBREVIATIONS…………………………………………………………....XI ABSTRAC T……………………………………………………………………………XIII CHAP TER 1: INTROD UCT ION 1.0 Back ground ……………………………………………………………………………1 1. 1 Problem statement …………………………………………………………………… . 2 1.2 Aim ……………………………………………………………………………………3 1.3 Objectives……………………………………………………………………………..3 1.4 Justification…………………………………………………………………………....3 1 .5 Hypothesis ……………………………………………………………………………..4 CHAP TER 2: LITERAT URE REV IEW 2.0 In cidenc e of breast cancer ……………………………………………………………..5 2.1 Risk factors of br east canc er ………………………………………………………….5 2.2.0 S ympt oms of breas t cance r …………………………………………………………6 2.2.1 Diagnosi s of breast canc er …………………………………………………………6 2.3.0 Typ es of bre as t cancer ……………………………………………………………...6 2.3.1 Gradin g/st a ging of breast cance r …………………………………………………..7 University of Ghana http://ugspace.ug.edu.gh VI 2.3.2 Prognostic indicators of breast cancer ……………………………………………...8 2.4.0 Treatm ent/ mana ge ment opti ons for breast cancer pati ents …………………………9 2.5.0 Estrogen and the o e strogen receptor ……………………………………………….. 9 2.5.1 A 36kil o Da lt on variant of the o es tro gen rec eptor - alpha………………………….. 11 2.5.2 Oe stro gen si gnali n g ………………………………………………………………. 12 2.5.3 Oe stro gen si gnali n g and breast canc er ……………………………………………. 12 2.5.4 Anti -o estrogens………………………………………..…………………………... 13 2.5.5 Cell migration and breast cancer metastasis…………. …………………………...13 2.6.0 Progeste rone and the pro gesteron e receptor ………………………………………14 2.6.1 Isoforms of the progesterone receptor……….. ………………………………… ...14 2. 6.2 Th e progesterone receptor and breast cancer ……………………………………...15 2.6.3 Hormone repl acem ent therap y (HRT) and breast cancer……….. ……………… ..15 2.7.0 The Human Epider mal Growth Factor Rec e ptor -2 (HER - 2/neu)…….……………16 2.7.1 HER -2/neu over ex pressi on and bre ast can ce r…………………………………… 17 2.8.0 Significanc e of ER, PR and HER - 2/neu in breast cancer…………………………18 2.9.0 Ax il lar y l ymph nodes as a possi ble alt ernati ve for ER, PR and HER -2/neu determ ination…………………………………………………………………………….20 2.9.1 Molecular subt ypes of breast cancer……………………………………………….24 2.9.2 Tripl e ne gati ve br e ast cancer (TNBC)……………………………………………..25 CHAP TER 3: MATER IA LS AN D METHODS 3.0 Study design …………………………………………………………………………27 3.1 Study site ……………………………………………………………………………27 3.2 Subjects/Target population …………………………………………………………28 University of Ghana http://ugspace.ug.edu.gh VII 3.3.0 Inclusion criteria …………………………………………………………………..28 3.3.1 Exclusion criteria ………………………………………………………………….28 3.4 Sampl e siz e determi nati on …………………………………………………………. . 29 3.5 Informed consent ……………………………………………………………………29 3.6.0 Use of control sam pl es …………………………………………………………….29 3.6 1 Positive controls ………………………………………………………………… ...29 3.6. 2 Negative controls ………………………………………………………………… 30 3.7.0 Sampl e prepar a tion ………………………………………………………………..30 3.7.1 Haematox yli n and eosin sta ining technique ………………………………………3 1 3.7.2 Imm unohis tochemi str y …………………………………………………………….31 3.7.3 Imm unohis tochemi cal staini ng procedu re …………………………………………32 3.7.4 Hydration of sections………………………………………………………………32 3.7.5 Epit ope retri eval……………………………………………………………………32 3.7.6 In cubati on with ant ibodi es a nd other reagents……………………………………..33 3.8.0 Staining rea cti on and reportin g crite ria for immunostains……………………….. . 34 3.9 Statistical analyses…………………………………………………………………...35 CHAP TER 4: RESULTS 4.0 Total number of cases………………………………………………………………36 4.1 Pathol ogical ch ara cte rist ics of cases……………………………………………… .37 4.2 Pathol ogical ch ara cte risti cs among tripl e n egative breast cancer cases……………43 CHAP TER 5: DIS C USS IO N AND CO NC LUS IO N 5.0 Comparison of ER, PR and HER - 2/neu…………………………………………….46 5.1 Conclusion…………………………………………………………………………..48 University of Ghana http://ugspace.ug.edu.gh VIII 5.2 Recommendation…………………………………………………………………….48 REFERENCES…………………………………………………………………………..49 APP END IX I …………………………………………………………………………….77 APP END IX I ……………………………………………………………………………79 APPENDIX III…………………………………………………………………………...80 University of Ghana http://ugspace.ug.edu.gh IX LIST OF TABLES Ta ble 1: Summ ar y of ER, PR and HER -2/neu resu lt s in primar y tum our and l ymph nodes ……………………………………………………………………..…... 37 Table 2: Subt ypes of breast canc er accordin g to receptor status ………………………..42 Table 3: Comparison of differen ces in ER, PR and HER -2/neu betwe en pr im ar y breast canc er and corr espondi ng l ymph nodes………………………………………………….44 University of Ghana http://ugspace.ug.edu.gh X LIST OF FIGURES Figu re 1a: Schematic repres entation of the human o estrogen receptor - alpha ………………………………………………………………………………... ........ 10 Figu re 1b: Sch ematic rep resentation of the human o estro gen receptor - beta ………………………………………………………………………………………10 Figu re 2: Schematic repr esentation of the human o estrogen rec eptor -alph a as compare d with the 36 kilo Dalton variant…………………………………………………………..11 Figu re 3: Micro graphs of control sli des…………………………………………….........39 Figu re 4: Micro graphs of o estrogen receptor positive slides……………………………. 40 Figu re 5 : Micrographs of progesterone receptor positive slides………………………… 40 Figu re 6 : Micro graphs of human epidermal gro wth factor rec eptor -2/neu posit ive slides……………………………………………………………………………………4 1 University of Ghana http://ugspace.ug.edu.gh XI LIST OF ABBREVIATIONS. AF1- Growth factor bind ing domain AF2- Li gand bindi n g do main Akt 1- Protein kinase B ASC O/CAP - American Societ y of Cli ni cal Oncolo g y/C oll ege of Am erican Pathol ogist s BRCA 1- Breast canc er gene 1 BRCA 2- Breast canc er gene 2 COOH- Carbox yl DNA- deox yribonu cleic acid ELIS A - Enz yme -li nked immunos orbent assa y ESR 1 - Oe strogen recepto r 1 ESR 2- Oe strogen recepto r 2 FIS H- Fluoresc en c e in situ h ybridi z ati on Ig - Imm uno globul in LNER- Oe stro gen recept or status in l ymph no de LNPR - Pro geste rone rec eptor status in l ymph nod e LN HER2 - HER2/neu sta tus in l ymph node n - Sampl e siz e MAPK- Mitogen acti vate d protein kinase PCR - pol yme rase chain reacti on PE LP - Proline, glut amat e and leucin e rich prot ein 1 P LN- Pe rcent a ge of l ym ph node invol vement University of Ghana http://ugspace.ug.edu.gh XII PTER - Oe strogen recept or status in primar y tum o ur PTPR - Progesterone rece ptor status in primar y tu mour PTHER 2 - HER2/Neu sta tus in primar y tum our SD - Standard deviation STAT- Signal transdu cer s and acti vators of tr ansc riptio n S IS H- Sil ver in sit u hyb ridiz ati on TN BC - Tripl e ne gati ve breast cance r University of Ghana http://ugspace.ug.edu.gh XIII ABSTRACT Introd u ction: Determi nati on of o estrogen recept or (ER), pro gest erone rec eptor (PR ) and human epiderm al growth facto r recepto r -2 (He r-2 /neu) has becom e pa rt of the dia gnost ic tests for breast canc er. R esult s of ER and PR are used to select bre ast canc er pati ents who are most likel y to respo nd to hormonal t her ap y where as Her -2/neu resul ts are us ed to select pati ents with inva sive bre ast cance r most likel y to respond to ta r get ed the rap y. Imm unohis tochemi c al (IHC) determi nati on of ER, PR and HER -2/neu is usuall y don e on the prim ar y br east can ce r tissue. Re -evalu ati on of ER, PR and HER -2/neu on metast ati c breast tum our is someti mes done when metastas is occurs. Th e ax il lar y l ym ph nodes ar e the most comm on sit e of metastasis of bre ast ca ncer. Cha ra cterist ics of prim ar y br east canc er ma y ch an ge whe n metastasis oc curs. This ma y affe ct the ex press ion of pro teins including ER, PR and HER -2/neu. Th e choi ce of the approp riate s ampl e for th e IHC determi nati on of ER, PR and HER -2/neu whe n metastasis occurs ma y ther efor e be cli nicall y relevant. Aim: The aim of thi s research was to compare the ex pressi on of ER, PR and the over- ex pressi on of HER - 2/neu betwe en prim ar y breast canc er and metastati c deposi ts in correspondi n g l ymph nodes in women with breast canc er at the Korle -Bu Teachin g Hospit al. Methodology: The stud y invol ved 54 archiv ed tissue blocks of prim ar y br east canc er tissue and corr espondi ng m etastati c deposi ts in l ymph nodes of wom e n with invasive breast canc er. The cas es were subm it ted to the Pathol og y Dep artment of The Korle- Bu Teachin g Hospit al betwe en J anuar y and Dec emb er 2009. Se cti ons wer e t aken from the tissue blocks and stained immunohi stochemi call y with anti bodies for ER, PR and HER - University of Ghana http://ugspace.ug.edu.gh XIV 2/neu. Stained secti ons were reported as eit her posi ti ve or negati ve ac cording to the guidelines of the Am erica n Societ y of Cli nical Oncolo g y/C oll e ge of Am erican Pathol ogist s (AS C O/CAP ). Results: Th e mean age ± SD of women diagnosed with breast canc er with l ymph node metastases was 49.89 ± 10.70 ye ars with a medi an age of 51 years. Th e percent a ge of retrieved ax il lar y l ymph nodes that were invol ve d with breast cance r cel l s ranged from 8% to 100%. Th e mean percent a ge of l ymph no des retriev ed from the ax illa was 57.87 with a median of 62.71 and a ran ge of 92. Abou t 22% (12 out of 54) of the cases had 100% l ymph nodes invo lved with breast can cer cell s and 3.7% (2 out of 54) had 8% of retrieved l ymph nod es in volved with breast can ce r cell s. About 20 % (11 out of 54) had gr ade I, 42.6% (23 out of 54) had gr ade II, and 37% (20 out of 54 ) had grade III bre ast tum ours respecti vel y. In the prim ar y br east canc er tum our, 31.5% (17 out of 54) of the cases wer e ER posi ti ve, 68.5% (37 out of 54) were ER negati ve, 25.9% (14 out of 54) were PR posi ti ve and 74.1% (40 out of 54) were PR negati ve. HER -2/neu was posi ti ve in 25.9% (14 out of 54 ) and negati ve in 74.1 % (40 out of 54) of the cases . In the correspondi n g l ymph no des, 33.3% (18 out of 54) wer e ER posit ive, 66.7% (3 6 out of 5 4 ) were ER negati ve, 29.6 % (16 out of 54) were PR positi ve, and 70.4% (38 out of 54) were PR negati ve . HER -2/neu was posi ti ve in 29.6% (16 out of 54) and negati ve in 70.4% (38 out of 54) of the cases. Th ere was 94. 4% and 92.5% con corda nce for ER and PR respecti vel y betw ee n prim ar y tum our and l ym ph nodes. The con co rdance rate fo r HER-2/neu was 96.3% . T here was no stati sti cal d ifferen ce (p > 0.05) in ER, PR and HER - 2/ neu between primar y breast cance r and metastat ic depos it s in l ymph nod es. University of Ghana http://ugspace.ug.edu.gh XV Conclusion: There wer e minor changes in the ex pressi on of ER, PR and HER -2/neu between prima r y bre ast tum our and metastatic deposi ts in l ymph nodes. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER 1: INTRODUCTION 1.0 Backgrou n d Bre ast canc er is the gro wth of malignant cell s in the breast. It is the most comm on malignan c y amon g wom en (23% of all can ce rs), and the second leadin g cause of canc er deaths in women (10. 9% of all canc ers), ex cludi ng non -melanoma skin cancers ( G LO BOCAN 2008 Ca ncer Fact She et ). Th e Internati onal Agenc y for Resea rch on Cancer (IARC ), based on the GLO BOCAN 200 8 report (Ferla y et al ., 2010), esti mated that in 2008, 1.38 million new cases of breast canc er wer e dia gnosed wo rldwide. The same repo rt indi cated th at, the incidenc e of br ea st cance r in the dev eloped co untries is higher ( 80 pe r 100,000) compared to that of developi ng countries ( 40 per 100,000). Howeve r, the mortali t y rate of bre ast canc er in th e developi n g countrie s is more than half (269,000) of th e esti mate d 458,000 deaths. This di ffer ence ma y be att ribute d to late s ta ge at diagnosi s and limi ted access to timel y stand ard treatm ents (Ber r y et al ., 2005). In Gh ana, bre ast canc er is the leading malignan c y in women (B adoe and Baako, 2000), accounti n g for 15.4 % of all malignanci es. The tre atm ent and / or mana gem e nt opti ons for breast cance r include su rger y and / o r radiot he rap y or chemot her ap y, hor monal therap y and tar geted the rap y. Th e use of hormon al ther ap y depends on th e presen c e of o estro gen recepto r (ER), and / or the pro gest erone recepto r (PR ) in the prim ar y breast tum our ( Ba rnes and Hanb y 2001 ; Haider et al ., 2001 ) . Br east canc er pati ents who se tum ours are HER- 2/neu posi ti ve will benefit from anti - HER - 2/neu targeted th er ap y (Dawood et al ., 2010) . University of Ghana http://ugspace.ug.edu.gh 2 1.1 Proble m stat e men t Assessm ent of ER, PR status and HER-2/ neu in breast cance r pati ents is usuall y made using the prim ar y bre ast canc er tissue (Zidan, 200 5; Idirisi nghe et al ., 2010 ). Fo r pati ents who have dev eloped dist ant metastases, ER, PR ex pressi on and HER -2/neu over ex pressi on are dete rmined on metastatic tum our deposi ts (Az am et al ., 2009; Hoefna gel et al ., 2010). Several studi es have suggest ed a differen ce in ER, PR ex pressi on and HER - 2/neu over- ex pressi on between prim ar y br east canc er tissue and met astatic tum our deposi ts (Tanne r et al ., 2001; Gipponi et al ., 2004 ; Azam et al ., 2009). Re c eptors ma y be lost due to tum our heterogen eit y (Nede r ga ard et al ., 1995; Azam et al ., 2009; Almendro and Fuster, 2011 ). At the Hist opathol og y Department of th e Korl e -Bu Tea ching Hospit al, Accr a, Ghan a, ex pressi on of ER, PR and the over-ex pressi on of HER 2/neu are deter mi ned using the prim ar y breast canc er tissue. Metastatic deposi ts in the l ymph nod es are us ed for receptor ex pressi on when the primar y br east canc er speci men is not avail able, or when the tissue chara cterist ics of th e pri mar y bre ast tum our is alt ered due to inad equate fix ati on. The prim ar y br east canc er tis sue ma y also have under gone nec rosis as a result of neo - adjuvant chemot herap y. This coul d lead to inapprop riate choice of ho rmone ther ap y and tar geted therap y i f there is a sign ificant differ ence in rec e ptor ex pressi on between prim ar y br east canc er and met astatic deposi ts in l ymph nodes. University of Ghana http://ugspace.ug.edu.gh 3 1.2 Aim The aim of thi s res ea r c h was to compar e the ex pressi on of ER, PR and the ov er - ex pressi on of HER - 2/neu betwe en prim ar y breast canc er and metastati c deposi ts in l ymph nodes in women with breast can cer at the Korle - Bu Teachin g Hosp it al. 1.3 Objectives 1. To determi ne the ex pres sion of ER, PR and HER - 2 / neu over-ex pressi on in prim ar y breast cance r tiss ue. 2. To det ermine the ex pr essi on of ER, PR and HE R - 2 / neu over -ex pressi on in metastatic deposi ts correspondi n g ax illar y in l ymph nod es . 3. To compare the ex pressi on of ER, PR an d HER -2/neu between prim ar y bre ast canc e r tissue and metastatic dep osit s in the l ymph nodes. 1.4 Justif ication The stud y proposal aim s at a bett er understandin g of the app ropriaten ess of the use of metastatic tum our deposi ts in the lymph nodes for the determinati on of ER, PR and HER - 2 / neu over-ex pressi on in women with breast cance r with ax illar y l ymph node invol vement . The in-depth stud y of those cas es might provide a bett er understandin g of the appropri ate tre atm en t in women with bre ast canc er. Th e outcom e of thi s stud y will provide rel evant info rmati on on the ex pressi on of ER, PR and the over ex pressi on of HER-2/neu in metastati c l ymph nodes in the ma nagement and/ or tre at ment of bre ast canc ers. University of Ghana http://ugspace.ug.edu.gh 4 1.5 Hypothesis There is no dif fer enc e in o estro gen rec ept or (ER), progesteron e receptor (PR ) ex pressi on and human epiderm al growth factor 2 (HER -2 / neu) over ex pressi on be tween prim ar y breast cance r and metast ati c deposi ts in l ymph no des. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER 2: LITERATURE REVIEW 2.0 Incid en ce of breast can cer. Bre ast canc er is a global healt h iss ue. It const it utes 23% of all can ce rs an d is the second leading (10.9% of all can cers) canc er worldwid e (G LOBOCA N 2008 Canc er Fact Sheet). More than half of deaths (269,000 out of 458,000 ) from breast can cer wor l dwide occu rs in developi ng countries (Ferla y et al . , 2008), inclu ding Ghan a (Wiredu and Armah, 2006). Studi es conducted by Bir it wum et al ., (2000 ) at the Korle - Bu Teachin g Ho spit al revealed that 12.8% of all admi ssi ons for mali gnant neopl asms wer e for bre a st ca ncer, and also most women report wit h advan ced disease to hospi tals (Arch ampong, 1977; Cle gg - Lampt e y et al ., 2007). 2.1 Risk factors of breast can cer. The gr eatest risk factor for developi ng br east can cer is gender (female ) an d the second is increasi n g age (Ya rdle y, 2000; American Canc er Societ y, 2011 -2012 ). Other risk facto rs include: inherit ed mut ations in the BRCA1 and BRCA2 gen es ( Struewin g et al ., 1997; American Can ce r Societ y, 2011 - 2012 ) , a person al or fami l y hist or y of breast cance r (Coldi tz et al ., 1993; J ohnson et al ., 1995; Yan g et al ., 1998; Lim et al ., 20 11; Americ an Cancer Societ y, 2011 - 20 12), high bre ast tissue densit y (Americ an Canc er Societ y, 2011 - 2012) , high - dose radiatio n to the chest wall ( Ng and Travis, 2009), and biops y - confirmed a t ypic al h ype rplasia (Ma rshall et al ., 1997; Ameri can Can cer Societ y, 201 1 - 2012). Some reproducti ve factors suc h as earl y men arch e an d late menop ause, neve r havin g given birth or givi ng birth for t he first time after age 30 are knoZn to increase a Zoman¶s risk University of Ghana http://ugspace.ug.edu.gh 6 of havin g breast can ce r (Antoni ou et al ., 2006; American Canc er Soci ety, 2011 -2012). Other facto rs that are as sociated with increased risks for breast can cer are modi fiable. These include: post menopausal obesit y, use of combi ned o estro gen and progesti n menopausal hormones, alcohol consum pti on, and ph ysical inacti vit y ( Am erican Canc er Societ y, 2011 - 2012 ). 2.2.0 Sympto ms of brea st can cer. The signs and s ympt oms of breast canc er include an y one or more of the fol lowing: lump in the breast, breast pain, ni pple dischar ge, nippl e retra cti on, breast skin ch anges like skin tethering or ulce rati on. 2.2.1 Diagnosis of breast can cer. Methods of assessment of a breast abno rmali t y include cli nical ex ami na ti on, imagin g , (mamm ogr aph y, ult ras ound, Ma gneti c Reso nance Ima gin g, Posit ron Emissi on Tomogr aph y) and fine needle aspir ate for cyt olo g y or biops y for hist ology. Th e diagnost ic te chnique em plo yed dep ends on the cli nical manifestatio n and the age of the woman. 2.3.0 Types of breast can cer. Breast canc er ma y aris e fro m the lobul es, ducts or an y of the connecti ve tissue components of the breast such as blood vessels. Ductal carcinom as are the most comm on t ypes of br east canc er. Rarel y, br east canc er ma y arise from the co nnecti ve tissue component of th e br ea st. Breast ca nce r ma y be confin ed to the lobul es, ducts or University of Ghana http://ugspace.ug.edu.gh 7 connecti ve tissue components of the mammar y gland (that is carcinom a in -sit u) or the canc er ma y spre ad to normal tissues insi de or outsi de of the mammary gland (that is invasive ca rcinom a). In vasive ductal car cinom a (IDC) is the most freque ntl y dia gnosed breast canc er in wom en worldwide (Har ris et al. 2000). Pagets¶ disease is a rare type of breast cance r that directl y invol ves the nippl e. 2.3.1 Gradin g/Stagin g of breast can cer. S tagin g and gradin g of cancers , includin g bre ast canc er , provide informati on that is used to predict the cli nical behaviour of the canc e r, establi sh appropriat e therapies and facil it ate ex chan ge of precise info rmati on betw een cli nicians (Sobi n, 2003; Cowherd, 2012). The Elst on -Elli s mo dificati on of the Scar f f- Bloom -R ichardson (S BR ) gradin g is used in the United States (Sim pson et al ., 2000). This gradin g s ystem co mbi nes nuclear gr ade, tubul e formation, and mitot ic rate. Each element is given a score of 1 to 3 (1 being the best and 3 bei ng t he worst). Th e lowest possi ble score (3 ) is given to well differenti at ed tum ours th at all fo rm tubul es and have low mitot ic rate (< 10/10 HPF). Th e highest possi ble s core is 9. A hist olo gical grade of III is assi gn ed an y t umour with a Notti ngham scor e of 8 or 9. The Tumour (tumour siz e and local growth) - No de (ex tent of l ymph nod e metastases) - Metastasis (occu rren ce of dist ant metastasis ) (TN M) classificati on s ystem describes the anatom ic al ex tent of breast cance r and all ows the groupin g of bre ast ca n cer int o stages (that is Sta ge I, Sta ge II, Stage III and Sta ge IV ). Sta ges III and IV ar e the adv anc ed stages of the cance r. Met astatic bre ast canc er des c ribes breast canc er that has spread from the breast to other parts of the bod y. Metastasis is usuall y to the ax il lar y l ymph nodes. University of Ghana http://ugspace.ug.edu.gh 8 The most comm on sit es of metastases from br eas t cancer are the lun gs, bones, liver and brain, accordin g to the Nati onal Can cer Insti tut e. Onc e metastas es ar e dete cted, the adopti on of s ystemi c the rap y is most l y pall iative (Mu ss et al ., 1991) whic h, acco rdin g to Andre et al ., (2004 ) ha s improved ove r time. Earl y det ecti on, opti mal sur ger y and adjuvant therap y are th e ke y str ate gies to improvi ng good pro gnosi s of breast canc er. 2.3.2 Prognostic in d icators of breast can cer. Th e Stage at dia gnosi s of breast canc er, l ymph node metastasis (Rack et al ., 2010), the hist ological t yp e of the prim ar y bre ast can cer (Na gao et al ., 2012), ho r mone rec eptor status and HER-2/neu over ex pressi on (Rampaul et al ., 2001), biol ogical tumour markers such as CEA (Hegg et al ., 1990) LDH and ALP and proli ferati on assa ys (Okta y et al ., 2012) have been reco gni z ed as pro gnost ic factors in breast canc er. Assess ment of these factors is important for cli nical management of breast canc er pati ents because these factors provide inform ati on for oncolo gist s for best therap euti c opti ons. It is stand ar d practi ce that hormone receptor status and HER -2/neu over ex pressi on are dete rmined prior to the comm encement of treatm ent for breas t cancer pati ents bec ause there has been an establi shed posi ti ve correl ati on between ER and PR with the degr ee of tum our differenti ati on (Mori et al ., 2001). Well differ enti ated bre ast ca rcinom as are mor e likel y to be ER and PR posi ti ve and have a bett er pro gno sis (Hilf et al ., 1980). University of Ghana http://ugspace.ug.edu.gh 9 2.4.0 Treatmen t and / or Manage men t option s for breast can cer patients. There ar e two groups of t reatm ent modalit ies fo r breast canc er; the lo cal an d the s ystemi c treatm ent opti on s . Lo cal treatm ent includes sur ger y and radiation. S yste mi c treatm ent includes chemot her ap y, hormone ther ap y and ta rgeted ther ap y. Breast canc er pati ents who have developed dist ant metastases usuall y under go s ystemi c therap y with chemot herap y, hormonal therap y and/or human epidermal growth factor receptor - 2(H ER 2/neu) tar geted th er ap y. Most breast cance r pati e nts at the Korle - Bu Tea ching Hospit al are given neo - adjuvant chemot herap y: About 60% of pati ents report to the Hospit al with advanc ed Stage of the disease ( Cle gg - Lampt e y et al ., 2007). Neo - adju vant endocrine therap y, ac cordi n g to Saigal et al., (2011), ma y be a good alt ern ati ve to neo - adjuvant chemot herap y for post menopausal women wit h ER - P osit ive breast ca ncer includin g locall y advanc ed breast cancer. ER, PR and HER -2/neu determi nati on has becom e part of the dia gnost ic t est s for breast canc er. Th e ER and PR result s help cli nicians i n selecti n g br east cance r pati ents most likel y to rec eive bene fit from hormon al ther a p y (Haid er et al ., 2001). HER -2/neu provides pro gnost ic in fo rmati on on recur renc e and survival (Mo ri et al ., 2002; Azam et al ., 2009). The pr esenc e of these ma rkers, acco rdi ng to Haider et al ., (200 1), Mori et al ., (2002) and Azam et al ., (2009) are related to the degree of the tum our diff e renti ati on . 2.5.0 Estrogen and the estrogen re cep tor. Estrogen is a lipophil ic hormone with low -mol e cular wei ght (app rox im atel y 300 g/m ol.) ( Hamm ond and Soule, 2004 ). It occurs natu rall y and pla ys a ke y rol e in t he regulation of sex ual and reprodu cti ve processes (Held ring et al . , 2007). 17 - o estr adiol (E2) is the most University of Ghana http://ugspace.ug.edu.gh 10 potent o estro gen produ ced in th e bod y, prob abl y bec ause th e o estr ogen receptor prefe renti all y binds it ov er o estriol (2x ) and o e strone (3x ) (Ruh et al ., 1973). E2 also binds to the o estrogen recepto r withi n the nucleus longe r than o estriol and oestrone (Gorski et al ., 1974; And erson et al ., 1975). Figure 1: a) Schema tic rep resen ta tion of the human o est rogen re cep to r -alp h a. b) Schema tic rep resen tation of the human o estrogen re cep to r -beta (Source: Held ring et al ., 2007 ) . There ar e two dif fer ent forms o f the o estro gen recepto r; Į and ȕ, each encoded b y a separat e gene ESR 1 and ESR 2 , respecti vel y. ER Į and ER ȕ are co-ex press ed in man y cell t ypes. Hormone- acti vate d o estro gen rec eptors fo r m dim ers . The dim ers ma y be ER Į (ĮĮ) or ER ȕ (ȕȕ) homodi mers or ER Įȕ (Įȕ) heterodimers. Result s of studi es co nducted b y Li et al ., (2004) suggests that ER Į is the functi onall y domi nant partner in the ER Įȕ (Įȕ) heterodim er . T h e centra l and most conserv ed domain of the ER is the DNA - bindi n g domain (DBD), which is invol ved in DNA rec ognit ion and bindi ng. Ligand bindi ng occurs at the mul ti functi onal COOH - te rminal of the ER (Nil sson et al ., 2001). ERĮ and ERȕ share a cons erved s tructure with six functi onal domains, A to F (Enmark et al . , 1997) . (F i gure s 1a and 1b) . The const it uti vely acti ve AF - 1 of the transcript ional functi onal domain is located at the NH2 te rminus of the ER and the liga nd - dependent University of Ghana http://ugspace.ug.edu.gh 11 AF -2 resides in the C OOH -term inal of the LBD (Nilss on et al ., 2001). ERȕ is homologous to ERĮ at the ligand-bindi n g do main (58%) and DNA - bindi ng domain (95%). The remaining domains are not well conserved (Enma rk et al ., 1997 ). ER Į is located on chromos ome 6 and it contains eight ex ons that encode a 66 -kDa full -len gth recepto r (Moria rt y et al ., 2006). E R ȕ is located on chromos ome 14 (Enmark et al ., 1997). 2.5.1 A 36k ilo Da lton varian t of the o estrogen recep tor-alp h a. S hi et al ., (2009) identified a 36kD a variant of the o estro gen rec eptor - alpha ( ER - Į) (66kDa) that ma y contrib ut e to tamoxi fen resis tan ce in ER -posi ti ve breast canc er pati ents ( Fowle r et al ., 2009; S hi et al ., 2009). A Stu d y conducted b y Zh an g et al ., (2011 ) concludes that ER - Į36 mediates non - genomi c oestro gen si gnali n g and is highl y ex pressed in ER-Ne gati v e breast canc er cell s. Figure 2: Schema tic re p resen tation of the human ERα as comp ar ed with the 36kDa varian t . (Source: Held ring at al., 2007). University of Ghana http://ugspace.ug.edu.gh 12 2.5.2 Oe strogen sign ali ng. In the cir culation, o estro gen is bound to sex stero id bindi ng globul in or to album in. Free acti ve o estro gen dif fuses across the cell membra ne and binds to a c ytopl asmi c receptor protein to form o estrogen -o estro gen rec eptor compl ex (McCart y, 197 7). Oestro gen - o estrogen receptor comp lex undergoes a t emper ature-dep endent alt e rati on in its ph ysic al properties prior to, or sim ult aneousl y with, its translocati on to the nucle us ( De Sombre , 1975) wher e it binds with deox yribonu cleic acid (DNA) and specific chromos omal proteins, alt erin g the patt ern of gene ex pressi on and the transcript io n of messenger ribonucleic acids (mRN As) (Maur er, 1974; Wil liams and Gorski, 1974 ). Studi es during the past few ye ars hav e demons trated that transcr ipt ional regulatin g functi ons of ER - Į66 in the nucleus can be inh ibi ted at th e same time as its ex tranucle ar ( memb rane) functi ons are bein g acti vated ( Levi n and Pietras, 2008 ). This ma y be useful in the pr ovisi on of new targets for the rapeuti c int erventi ons ( Gir ett i et al ., 2008). 2.5.3 Oestrogen sign ali ng and breast can cer . The o estro gen rec eptor, which has no trans mem brane and kinas e domai ns is known to ini ti ate E2 rapid signali n g with the form ati on of compl ex es that acti vates the MAPK1/3 and AKT1 in breast can cer cell s (Song and Santen, 2006). Other protein compl ex es that are required fo r the ini ti ati on of the rapid acti ons of oestro gen include ES R1, CSK, SHC1 and PE LP 1. PE LP 1 protein ex pressi on is an independent pro gnost ic predi ctor of shorter breast canc er sp ecific su rvival and dise ase fre e s urvival in bre ast cance r and its elevat ed ex pressi on is posi ti vel y associated with marke rs of poor outcome (Habash y et al ., 2010) . University of Ghana http://ugspace.ug.edu.gh 13 In ER- posi ti ve bre ast ca ncer pa ti ents, PE LP 1 ap pears to have a potential appli cati on in assessing the i r cli nical o utcome. 2.5.4 Anti-o estrog en s Anti -o estro gens, desi gne d to block ER - Į, are widel y and effe cti vel y us ed cli nicall y in the treatment of breast cancer (Early Breast Cancer Trialists¶ Collaborative Group 2005; Clarke, 2006). The re ar e two classes of anti -o es trogens: Sele cti ve Oe str ogen Re ceptor Modul ators (SER Ms) which ar e used fo r estro gen inhi bit ion or suppr es sion to prev ent recur renc e of tum ours, and aromatase i nhibi tors presc ribed after pri mar y tre atm ent (sur ger y, ch emoradiatio n) to prevent recurr enc e of breast canc er in post menopausal women. Tamox ifen is th e most comm on SERM used. Anastr az ole - oral (Arim idex ) is the most comm on aromatase inhi bit or used at the Korle - Bu Tea chin g Hospit al (personal comm unicati on with Mr. Clegg - Lampt e y , Head of Department Sur ger y ) . Ot her aromatase inhi bit ors used are , l etro z ole and ex emestane . The over all ef fects of anti - estrogens on breast can cer cell s include tum our shrinkage ( Dh ingra , 1999), a decr ease in the numbers of cell s in th e S - phase of the cell c ycle ( D alvai an d B ystrick y , 2010 ) and t he induction of cell ular apoptos is ( Riggi ns et al ., 2005). 2.5.5 Cell migration and breast can cer metas ta sis C ell migrati on is a key fi rst step in the met astasis of bre ast can cer . Mechanism s underl yin g the eff ects of o estrogen on metastasis ma y be diffe rent from t hat of tum our regressi on. Inhibi ti on of o estrogen s ynth esis in breast canc er result s in tumour regressi on and reducti on of new metastasis . Estrogen (E2) int eracts with ESR 1 and several othe r recepto rs to facil it ate E2 induced cell mi gr ati on. The pathwa ys that invol ve thes e University of Ghana http://ugspace.ug.edu.gh 14 recepto rs have been studi ed to develop models tha t will inhi bit tum our cell migrati on and metastasis (Li et al ., 2010). 2.6.0 Progest eron e and Prog este ron e recep to r. P rogeste rone is a C-21 steroid hormone. It is lipophil ic and diffuses across the cell membrane of its ta r get c ell wher e it binds to a cyt oplasmi c rec eptor ( Mc C art y, 1977 ) to form a pro gesteron e- pro geste rone rec eptor comp lex . Progesterone - pro ges terone recepto r compl ex is translocated int o the nucleus ( De So mbre, 1975 ) wher e it bi nds to specific proteins in the nucl eus of its target cell and alt e rs the ex pressi on of genes in the tar get cell ( Maure r and Chalk y, 1974; Willi ams and Gorski, 1974 ). The progester one rec eptor is also known an NR3C 3. In humans, PR is encode d by a single gene locate d on the long arm of chromoso me 11 (11q22) (Law et al ., 1987). 2.6.1 Isof orms of the Progesteron e re cep tor. There are two main nu cl ear isoforms of the hum an PR; A and B (Grah a m et al ., 1995). Aside the lack of ami n o - termi nal 164 ami no acids that form the thi rd transacti vati on dom ai n of PRA, both isoforms are structurall y identical (Kastner et al ., 1990). Studi es conducted b y Mote et al ., (2002) rev ealed that PRA predomi nan ce was evi dent in a high proportion of ductal ca rcinom as in sit u (DC IS ) and invasive breast lesions. Over abundanc e of PRA is rel ated to Tamox ifen resis t ance (Hopp et al ., 2004) , while ex cess producti on of PRB is ass ociated with bre ast can ce r risk (Osborn e et al ., 20 05) and poore r outcome of ch emot herap y. Some protein produc ts from PR target genes are associated with mammar y gl and an d breast canc er develop ment, including the t rans criptio n factors University of Ghana http://ugspace.ug.edu.gh 15 STAT5A and C/EBPȕ (Cork et al ., 2008). Ther e ex ist s a cytopl asmi c PR protein, PRC which lacks a full length DNA bindi ng dom ain (W ei et al ., 1990). PRC in humans has been proposed to pla y a potential ph ysiol ogi cal ro le in pre gnanc y ( Condon et al ., 2006). 2.6.2 The P rog este ron e rec ep tor and breast can cer . Accordin g to Cork et al ., (2008), there ex ist other variants of PR with loss of specific functi onal domains which ma y alt e r the progesti n responsi veness of a tissue and contribut e to the abnor mal growth asso ciated wi th breast canc er. PR is an estrogen responsi ve gene henc e PR posi ti vit y indi cat es not onl y th e pres ence of ER but also a functi oning ER pathwa y (Horwitz and McGuire , 1975; Osborne et al ., 2005). Some PR negati ve tum ours (loss of PR due to HER - 2 ove r ex pres sion which down regulates the PR gen e) corr elate with resp onse to aromatas e inhi bit ors but not SERM s. This could be due to the ex ist ence of crosstalk betwe en ER, PR and HER - 2/neu (Cui et al ., 2005; Osborne et al ., 2005). Pati ents with ER - posi ti ve/P R - posi ti ve tum o u rs have a bett er prognosi s than pati ents with ER - posi ti ve/P R - negati ve tum o u rs, who have a bett er pro gnosi s than pati ents with ER - negati ve/P R - negati ve tum o u rs (Gown et al ., 2008; Lin et al ., 2012). 2.6.3 Hormon e rep lace men t therap y (HRT) an d breast can cer . Progeste rone throu gh the PR is of signific anc e in the developm ent of breast canc er (Stahlber g et al ., 2004). The Nati onal Tox icology Pro gr am in its Decem ber 2002 press releas e declar ed o estro gen as a car cinogen. The re is increased risk of breast c an cer in women on HRT with combi ned o estro gen - p ro gesterone compar ed to o estrogen alone ( Campa gnoli et al ., 2005). University of Ghana http://ugspace.ug.edu.gh 16 2.7.0 The Human Epider mal Growth Factor Receptor-2 Human Epidermal Gro wth Factor Re ceptor -2 (HER -2/neu) is a member of the Erythrobl asti c Le uk emi a Vir al Onco gen e (Er bB) protein fami l y. The HER -2/neu , encoded b y the ERB B2 gen e is a proto -onco gen e located at 17q12 - 21.32 ( Popescu et al ., 1989). Transm embrane Her - 2/neu mol ecules are ex pressed as ina cti ve monom ers on the cell surf ace . Li gand bind ing result s in dim eriz ati on of the monom e rs (Alro y and Yarden , 1997; Schlessinger, 2000 ) leadin g to the phospho r ylation of specifi c t yr os ine residues in the receptor c ytopl asmi c tails (Schlessi nger, 2000 ). Subsequent acti vati on of signal transducti on pathwa ys le ads to cell growth and dif ferenti ati on ( Ol a yio ye , 2001). In about 10 % to 40 % of prim ar y breast can cers, the HER -2/neu gene is ampl ified, with subsequent over ex pressi on of the HER -2/neu protein and is associated with more aggressi ve diseas e (Salmon et al ., 1987; Rubin and Yarden, 2001; Paluch - S him on et al ., 2005). Several studi es have been don e to corr e late HER - 2/neu gene am pli ficati on and protein over ex pressi on with disease outcome. HER - 2/neu protein ove r ex pressi on by immunohi stochemi str y (IHC ) has been found to have signific ant correl ati on with disease outcome on univariate anal ysis but has fail ed to demons trate indepen dent predictive status for immuno rea cti vit y on mul ti variate anal ys is (No gu chi et al ., 199 2; Hartmann et al ., 1994). HER - 2/neu gene ampl ific ati on and HER - 2 protein over ex pre ssi on has been observed in man y hum a n cance rs including end ometrial ( Hetz el et al ., 1992), uterine cervix (Mitra et al ., 1994 ), head and neck ( Beckh a rdt et al ., 1995 ), bre ast (Kaptain et al ., 2001), bladder (Elt z e et al ., 2005), colon ( Schuell et al ., 2006), oesophageal ( Reichelt et al ., 2007), and gastric ( G ravalos et al., 2008). University of Ghana http://ugspace.ug.edu.gh 17 2.7.1 HER-2/neu over exp ressi on and breast can cer. Her-2/neu is ex press ed at low levels in normal non -neoplasti c epit heli a, i ncludi ng br east duct epit heli um, and is over -ex pressed in bre ast canc ers (Slamon et al ., 1987; Paluch - S him on et al ., 2005). B reast canc ers can hav e up t o 25 - 50 copies of t he HE R - 2/neu gene, and up to 40 - 100 fold increas e in HER - 2/neu protein result ing in 2 mill ion receptors ex pressed at the tum o u r cell surf ace ( K all io niemi et al ., 1992). The HER - 2 pathwa y invol ves a compl ex biologic al network comprisi ng of ligands, comi ng from outsi de the cell that bind to memb ra ne receptors, a numb er of protein kinas es tr ansmi tt ing the si gnal to the nucleus and transcript ion factors regulat ing genes that aff ect various cell ular functi ons (Cit ri and Yarden, 2006). The HER - 2/ne u signali ng pathw a y, as wel l as the ER signali n g pathwa y are t he domi nant drivers of cell proli ferati on and su rvival in about 85% of all breast canc ers ( Gutierrez and Schiff , 2 011 ). Among the HER fami l y membe rs (HER1 to 4), HER - 2 is the domi nant t yrosine kinase in br east cance r. HER - 2 does not have a ligand and rel ies on hete rodimeriz ati on with another fami l y memb er or homodi meriz ati on with itself when ex pressed at ver y high l evels to be acti vated (Cit ri and Yarden, 2006). HE R - 2 ex ists in an open conformation ex posi ng its dim eriz ati on domains making it the di meriz ati on partner of ch oice amo n g the fami l y m embers. When HER- 2/neu is over - ex pressed, t yrosine kinase is const it uti vel y acti vat ed ( Moasser et al ., 2001), result ing in mitogenic transducti on and po or pro gnosi s ( Zh an g et al ., 2004). HER - 2/neu over ex pressi on has been associated with sig nificantl y shorten ed disease - f ree and overall survival (Sesh ad ri et al ., 1993). The ins uli n - li ke - gro wth factor receptor 1, can compl ex with HER - 2 leadin g to HER - 2 acti vati on (Nahta et al ., 2005). Oestro gen, working via the non - ge nomi c acti vit y of ER outsi d e the nucleus has been shown to University of Ghana http://ugspace.ug.edu.gh 18 acti vate HER-2 si gnali n g (Shou et al ., 2004). P95, an aber rant form of the HER -2 has been found in some br e ast canc ers (Molina et al ., 2002; Scalt riti et al ., 2007). P95 is related to the ex tent of l ymph node invol ve ment an d is enhan ced in nodal tissue suggesti n g an important role as a ma rker or caus e in breast canc er metast asis (Molina et al ., 2002). P95 is const ituti vel y acti ve since the ex ternal domain of these receptors acts as an inhi bit or unti l the y ar e bound b y li gand . P95 lacks the ex ternal domain of the HER -2, so it is not detected by anti bodies that target the ex ternal domain of HER -2. P95 can cause resis tance to tr anstuz umab (Carolina et al ., 2011). 2.8.0 Signif icance of ER, PR and HER -2/neu in breast can ce r. W om en with breast cancer, as part of their treatm ent and / or mana ge ment, undergo surger y and/or radiot her ap y, chemot her ap y or ho rmone/endocrin e therap y depending on the age of the indi vidua l and stage of the br eas t cance r at dia gnosi s. The addit ion of radiot he rap y to sur ge r y and adjuvant s ystemi c treatm ent redu ces th e risk of an y rec ur renc e of breast can cer (Whelan et al ., 2000), and those who suffer a recurr enc e usuall y do not have aggressi ve liver or central nervous s ystem invol vement ( Maki and Grossm an , 2000 ) mainl y as a result of an incre ase in loco - re gion al control. P resence of ER and PR in breast canc er tissues as determi ned by immunohi stochemi str y correl ates well with res ponse to endo crine th er ap y and chemot he rap y ( Earl y Br east Cancer Triali sts ' Coll ab orati ve Group, 1998 ; Barnes and Hanb y 2001; Haider et al ., 2001), and provid e s pr ognost ic inform ati on on recu rren ce and survi val since their ex pressi on is related to the degre e of the tum our differenti ati on (M akki and Grossm an, 2000). Studi es conducte d in the United States show that a bout 70 - 80 % of breast cance rs University of Ghana http://ugspace.ug.edu.gh 19 are oestro gen rec eptor -p osit ive (Keen and David son, 2003; Gown, 2008 ) . These tum o u rs tend to grow more slow l y, are bett e r diff erenti at ed, and are associated with a sl ightl y bett er ov erall pro gnosi s (Clark, 2000). Approx im atel y 30% of breast tu mours ar e ER - Ne gati ve (Lac roix et al ., 2004; Sim pson et al ., 2005). These cance rs are gen erall y mor e aggressi ve than ER -p osit ive breast canc ers. Appr ox im atel y 40% of bre ast canc ers ar e ER posi ti ve and PR posi ti ve (Mar golese et al ., 2000). HER2/neu status is known to be a pro gnost ic as well as pr edictive mark er in both node - negati ve (Andruli s et al . , 1998) and node-posi ti ve bre ast can ce r pati ent s (Muss et al ., 1994). The most succ ess ful ex ampl e of tar geted therap y in metast ati c bre a st cance r is the target t ing of hum an epid ermal growth factor rece ptor - 2 (HER2) b y trastu z umab ( Barton and Swanton 2011). Tra stuz umab is a monoclonal anti bod y that t ar gets th e ex tra cell ula r domain of the HER -2/ne u protein. Trastuz umab does not block autophos phor ylation of HER-2, howeve r it inhi bit s HER -2 downstream signali n g (Nahta et al ., 2006). Other mechanism s of acti on of tr astuz umab ma y include disrupti on of the HER -2/S rc int eracti on, int ernali z ati on and d own regulatio n of the rec eptor and enhanc ement of anti bod y mediated c ytot o x icit y. Studi es conducted by Dawood et al. , (2010) reveal ed that transtuz umab improved prognosi s in HER -2/n eu posi ti ve breast cance r women. About twent y- five per cent of metastatic br east canc er pati e nts rec eivi ng first line tre atm ent respond to trastuz umab alone (Vo gel et al ., 2002) . Several cli nical trail s (J oensuu et al ., 2006; Perez et al ., 2007; Smi th et al ., 2007) have shown that trastuz umab in combi nati on with different chemot her ap y has si gnific antl y improved disease free survi val and overall survival wit h reducti ons in the risk of recurr ence o f breast canc er ran ging from 40 -50%. University of Ghana http://ugspace.ug.edu.gh 20 Studi es conducted b y Makki an d Grossman (200 0) conclud ed that ER/ P R status can help the radiol o gist in detec ti ng metas tatic breast cancer: pati ents with ER -posi ti ve/P R - posi ti ve (ER+/P R +) brea st cancer tend to develo p bone metastases; whiles pati ents with ER -negati ve/P R -ne gati ve (ER -/P R -) tum ours tend to develop metastases in the brain. This will help the cli nician to esti mate the likeli hood of metastases to various organ s ystems, as well as to potentiall y tar get therap y. Molecules invol ved with ER signali ng pathwa y to a large ex tent influence endocrin e responsi veness of bre ast cance r (Rastelli and Crispino , 2008 ). C1orf64 or ER -related factor (ERR F), an ER si gnali ng mol e cule, ma y be targeted for the rap euti c purposes ( Su et al ., 2012). 2.9.0 Axillary lymp h nod es as a possib le altern ative for ER, PR and HER-2/neu deter min ation. Endocrine ther ap y and targeted the rap y fo r bre a st canc er pati ents are based on the hormone receptor status and the ove r ex pressi o n of HER -2/neu of the prim ar y bre ast canc er tissue (Lowe r et al ., 2005; Guarn eri et al ., 2008; Rack et al ., 2010). Tumour deposi ts in the met astatic sit e are us ed to d etermi n e the response to endocri ne ther ap y and targeted th erap y when metastasis occu rs (Daw ood, 2010). It is somet im es howeve r difficult to access meta static tum our (Aktas et al ., 2011). Breast can cer comm onl y spreads throu gh l ymph nodes. Sevent y- five perc e nt of all breast tum our and gre ater th an ninet y- five perc ent of all larger bre ast tum our gen erate l ymph node metast ases (Har rell et al ., 2006). Presence of ax illar y l ymph node meta stasis , accordin g to Rack et al ., (2010), is the most important predictor for dis ease -fr ee and ov erall survival of bre ast cance r University of Ghana http://ugspace.ug.edu.gh 21 pati ents. Harr el et al ., in 2006 developed met astas is models using ZsGr een labeled MCF - 7 and T47D human breas t canc er cell s in nude mic e. The y obs erved that pro li ferati on was higher in l ymph nodes t han in pri mar y bre ast tum our. Harr ell et al ., observed also that unli ke in the prim a r y tu mour wher e hi gh l evels of ER are do wn -r e gulate d by E2, the re is partial fail ur e of ER down -re gulation in l ymp h nodes associat ed wit h reduc ed PR ex pressi on. The pres enc e of ho rmo ne rec eptors and HER -2/neu in l ym ph nodes ma y therefor e be important i n the endoc rine and tar geted ther ap y tre atm ent of bre ast can cer pati ents with posi ti ve l ymph node metastasis . Accordin g to Harr el et al ., in the l ymp h node microenvironment, ER ma y howev er be dysfun cti onal and per haps hormone resis tant. S everal studi es have be en condu cted to compar e the ex pressi on of ER, PR and HER - 2/neu over ex pressi on in prim ar y breast canc er tissue and tissue from diffe rent metastatic sit es. There howev er ex ist some d iffer ences in the result s from the various studi es. Factors su ggested to be responsi ble for the chan ge in hormon e receptor st atus and HER - 2/neu in metastatic bre as t canc er in clude: tum o u r hetero gen eit y (Neder gaar d et al ., 1995; Azam et al ., 2009; Arslan et al ., 2011), clonal selecti on of tum o u r cell subpopulations , gen eti c inst abil it y of tum o u r cell s (Edge rton et al ., 2003; Prat and Perou, 2009) , local or systemi c tre atm ents, the time int erval between pr im ar y tum o u r and metas tasis, receptor status determi n ati on techniques (Prat and Perou, 2009) , and the sit e of metasta sis (Arslan et al ., 2011). Unmekit a et al ., (1998) and Bo gina et al ., (2011) in their stud y obse rved that loss of PR in recurr ent and metastatic (site not stated) breast cancer was frequent and correlated with a worse pro gnosi s ; a cha nge in ER was infrequen t and a change in HER -2/neu was rare. University of Ghana http://ugspace.ug.edu.gh 22 Bo gina et al ., 2011, ther efore su ggested the reas sessm ent of HER -2/neu with both IHC and FIS H in metastatic breast can cer wh en there is a change in HER-2/neu between the prim ar y tum our and Met astatic tumour. Studi es conducted by Lower et al in 2005 showed a corr elation betwe en ER and PR between prim a r y and Metastatic (site not stated) breast can cer . Out of the 200 cases anal yz ed for ER , 39(19. 5%) were po sit ive in th e prim ar y tum our and negati ve in the metastatic tum our. 21 (1 0.5%) cases had ER negati ve prim ar y tum ours and ER posi ti ve metastatic tum ours. Ther e was discordan ce, (30 %), betwe en prim ar y an d metastatic ER status. Tumo urs from 68 of 173 (39.3%) showed discordanc e for PR. Result s of studi es of Gan cber g et al ., (200 2) did not support routine dete rminati on of HER - 2 on metastatic sit es, particularl y when FIS H result s from the pr im ar y tum our are avail a ble. Guarn eri et al ., (2008) and Sari et al ., (2011) howev er co ncluded in their stud y t hat biops y of metastatic bre ast can cer is recomm ended, if fea si ble with minim al invasiveness be cause treatm ent opti ons might chan ge fo r a signi ficant proportion of breast canc er pati ents with metastasis (Guarn eri et al ., 2008; Sari et al ., 2011). Broom et al ., in 2009 fou nd that signific ant discor dance ex it s for hormone status between prim ar y and metastatic breast can cer with a freq uent loss of PR. Broom et al . showed 17.7% and 37.3 % (n =10 0) discordan ce betwe en prim ar y and metastatic breast canc er for ER and PR resp ecti vel y. 9.7% of prim ar y tum o urs chan gin g from ER - p osit ive to ER - negati ve and 8.0% changin g f rom ER - n e gati ve to ER - posi ti ve. No signifi cant disc ordanc e for Her - 2/ne u was found. Simmons et al ., (2009) also s how e d the presen ce of subst anti al discordance in hormone receptor s between prim ar y br ea st cancer and metastases, which led to alt ered man a gement on 20% of cas es. University of Ghana http://ugspace.ug.edu.gh 23 Aktas et al ., in 2011 compared ER and PR ex pressi on in prim ar y breast cance r with circulatin g tum our cell s. Breast canc er pati ents with ER -posi ti ve and PR posit ive tum ours had cir culating tum our cell s (CTCs ) which were ER -ne gati ve and PR - ne gat ive. Treatm ent based on the ER and PR status of the prim ar y bre ast cance r m a y th ere fore be ineffe cti ve in met astatic disease. Holdaw a y et al. in 1983 show ed con cor dance of 46 % between primar y and me tastati c breast can cer. Si x out of nine of the case s that wer e ER - posi ti ve in the prim ar y t umour were ER - ne gati ve in the metastatic disease. Four out of five PR - po sit ive cases in the prim ar y tum our wer e PR - negati ve in the met astatic tum our. Nine out of nineteen ER - posi ti ve and three out of fifteen PR - posi ti ve cases in the prim ar y tum our were ne gati ve in the metastatic tum our respecti vel y. Holdaw a y et al. attributed some of the chan ges in recepto r ex pressi on to treatm ent that the pati ents had rec eived during the cours e of the disease. Azam et al. in 2009 compared ER and P R ex pressi on in prim ar y breast cance r with correspondi n g metasta ses in l ymph nodes. Abo ut 28 % (28 out of 100 ) pati ents had ER - posi ti ve prim ar y tum ours. ER ex pressi on in the correspondi n g l ymph nodes was 25%. The perc enta ge of PR posi ti vit y in the prim ar y br ea st tum our was reduced from 28% to 22 %. HER - 2/neu ex pressi on howeve r, remained al most the same b etween the primar y tum our and tum our metastas es. With regards to the abo ve mentioned studi es, t here s eem to be a ch an ge in receptor ex pressi on in the metastatic disease. Small as thi s proportion ma y be, it is necessa r y to identif y women with me tastati c br e ast can cer wh o belong to thi s group, since survival time ma y depend on rec e ptor ex pressi on in the me tastati c tum our (Holdaw a y et al ., 1983; Azam et al ., 2009; Aktas et al ., 2011 ). There ex ist phenot ypic ch an ges be tween prim ar y and metastatic bre ast ca nce r (J acot et al ., 2011). This has been h ypothes iz ed to be the University of Ghana http://ugspace.ug.edu.gh 24 cause of di fficult y faced in the treatm ent and / or mana gement of breast can cer pati ents with metastasis . Tre atm ent (neo adjuvant, sur ge r y, radiation, chemot her ap y, adjuvant therap y and HER -2 -tar geted the rap y) ma y mod if y ER, PR and HER -2 /neu betwe en prim ar y and metastatic di sease ( Liu et al ., 2012 ). 2.9.1 Molecu lar sub types of breast can cer Growin g evidenc e su gge sts that b reast canc er is a hetero gen eous disease defined b y ER, PR, and HER - 2 /neu ex p ressi on with distinct a e ti ologic al pathw a ys and pro gnoses (Kwan et al ., 2009). Breast can cer can be sub t yp ed ac c ording to h or mone rece ptor status and HER- 2/neu status (Bau er et al ., 2007): ER+ and/o r PR+, HER - 2/neu ± (lu mi nal A), ER+ and/or PR+, HER - 2/neu + (lumi nal B), ER - , PR - , HER - 2/neu - (Bas al or Tripl e negati ve) and ER - , PR - , HER - 2/neu + (HER - 2 over ex pressi ng). Consi derabl e diffe rences ex ist in survival, demographic and tum our char acterist ic s between ER, PR and HER - 2 bre ast canc er subt ypes (Parise et al ., 2009). Parise et al ., 2009 recomm ended reporting bre ast canc er as an ER/ P R /HER - 2 subt ype and do cume nti ng pre cisel y demograp hic and tum our chara cterist ics. Some of the result s of studi es co nducted b y Bau er et al ., (2010) include the following; ER neg ati vit y app ear to be a stron ger pr edictor of poor su rvival than HER - 2 posi ti vit y, HER - 2 posi ti vit y did not alwa ys tra nslate to worse survival as noted when compared tripl e posi ti ve subt ype to tripl e ne gati v e subt ype, th e hetero gen eit y of the hi gh risk cate gor y was most evident in the ER/P R /HER - 2 subt yp e with four or more posi ti ve ax il lar y l ymph nodes. Ba uer et al ., therefore reco mm ends the correlation of ER/ P R /HER - 2 subt ype with mol ecula r classificati on. Accordin g to Troeste r and Swift - S calan, (2009), regio nal vari ati on s in bre ast canc er subt ypes contr ibut e to racial disparities of the disease. University of Ghana http://ugspace.ug.edu.gh 25 2.9.2 Triple negative breast can ce rs. Tripl e ne gati ve br east canc ers are bre ast can cers th at ar e immunoh ist ochemi call y classified as ER negati v e, PR negati ve and HER -2/neu ne gati ve. T riple negati ve breast canc er (TN BC ) const it ute about 15% of all breast cancers worldwid e ( Lar a -Medina et al ., 2011). Bla ck women hav e a high er inciden ce of tr ipl e negati ve bre ast can c ers than white women re gardless of age and bod y mass ind ex (Stead et al ., 2009; Rais et al ., 2012 ). Studi es have shown that thi s subt ype of br east can cer oc curs prim aril y in younge r women of African Americ an or Hispanic descent (Grif fiths and Olin, 2012; Dawood, 2010). Result s of studi es condu cted b y Stark et al . in 20 10, showed that amon g t he 75 cases of Ghanaian women with br east canc er at The Komf o Anok ye Teachin g Hos pit al, 61 (81%) had tripl e ne gati ve bre ast canc er . In th e same stud y, 26% (n=581 ) African Americans and 16% (n=1008) Whit e Americans had tripl e ne gati ve breast can ce r. In a sim il ar stud y conducted b y Adisa et al . (2012) in Ni geria, 65% (n=17) of Nigeria n wo men with bre ast canc er were tripl e ne gati ve an d had hi gh gr ade ca ncers (100 % grad e III) . Tripl e ne gati ve breast canc er is char ac terist icall y ag gressi ve with high recu rren ce, metastatic, and mortali t y rates (Brown et al ., 2008; Dawood, 2010) but ini ti all y respond s to tradit ional s ystemi c c ytot ox ic chem otherap y (Dawood, 2010 ). Potential future therap ies for TNBC include tar geted mol e cular str ate gie s includin g po l y ADP ribose pol yme ra se (PARP ) an d epidermal growth factor recepto r (EG FR ) inhi bit ors and anti an giogenic agents (Duff y et al ., 2012; Griffit hs and Olin, 2012). Over ex pre ssi on of EGFR has bee n shown to be associated with TN BC and poor survi val in Tunisi an women (Kall el et al ., 2012). S ystemi c evaluation of EGFR in breast c ance r could benefit t riple negati ve subgroup University of Ghana http://ugspace.ug.edu.gh 26 from EG FR tar geti ng agents. Women with metas tatic tripl e -ne gati ve br ea st canc ers have a much shorter medi an time from relaps e to death (Dent et al ., 2007). 19p13.1 is the first triple -ne gati ve -speci fic breas t cancer risk locus and the first locus specific to a hist ologic al subt ype de fined b y ER , PR , and HER2 to be identified (Stephens et al ., 2012). Result s of studi es from three loc ati ons in Nigeria and one loc ati on in Senegal publi shed in the Scienc e in Africa ( Olopade, 2005) rev eal th at bre ast can cer in Africa n women ma y be geneti call y diffe rent from breast can cer in Whit e women. This, accordi ng to the stud y author ma y ex plain the absence of the mol ecula r targets that form the basis of man y standard therapies: 80% of breast can cer in Cauc asian women have ERs, 23% of breast canc er in Afric an wome n ar e ER+. Breast can cer in Cauc asian women are more likel y to be HER-2/neu + (23%) th an breast can cer in African women (19% ). Hence the worse prognosi s of bre ast can cer in Afric an women a s compared to the Whites ( Science in Africa, 2005 ). University of Ghana http://ugspace.ug.edu.gh 27 CHAPTER 3: MATERIALS AND METHODS 3.0 Study design. The stud y was a retrosp ecti ve stud y which invol ved the use of archiv ed formalin fix ed, paraf fin wax embedd ed prim ar y bre ast canc er tissue blocks of women, and their correspondi n g ax il lar y l ymph node metastase s su bmi tt ed to the Pathol ogy Department of The Universit y of Gh a na Medical School (U GMS ) , Korle- Bu, betw e en J anuar y and Decemb er 2009. 3.1 Study site. This stud y was carri ed out between the mont hs of Ja n uar y and Decemb er 2012 at The Pathol og y Depa rtment , of The Korle ±Bu Tea chin g Hospit al (KBTH) , Acc ra, Ghan a. The KBTH, situated in the nation¶s capital Accra is the leading tertiary hospital and the major refe rral cent re in the countr y. It also serves as th e leading teachin g hospi tal of The Universit y of Ghan a Me dical School (UGMS ). The Pathol og y Depa rtment is the biggest terti ar y care centr e in the count r y. Mor e than 75 % of the tot al number of breast canc er cases s een in the count r y are proc essed th rou gh t his department. Thus the demo graphics of the pati en ts that were tested in this stud y wer e not limit ed to a specific social group. The pati ents in this study ori ginated from vario us social and ethni c groups as well as geo graphic all y dist inct ar eas from each region of Ghana. University of Ghana http://ugspace.ug.edu.gh 28 3.2 Subjects/target pop u lat ion. S ubjects for the stud y were female pati ents with invasive breast cance r with axil lar y l ymph node metastas es obtained from the hist opathol og y dat a entr y book (log book) for the ye ar 2009. 3.3.0 Inclu sion criteria. Bre ast specim en of women diagnosed with invasive breast can cer wi th l ymph node metastasis subm it ted to the Depa rtment of Pathol og y of the Ko rle - Bu Tea ching Hospit al between J anuar y and December, 2009. 3.3.1 Exclu sion criteria. 1 - Cases of breast cance r without ax illar y l ymph n ode invol veme nt . 2 - All cases that fitted into the above mentioned inclusi on criteria but whose prim ar y breast cance r was eit her; A- Not avail able or B- Not prop erl y fix ed or C- Had features of tissue necrosis. University of Ghana http://ugspace.ug.edu.gh 29 3.4 Sample size de ter mi n ation. From the formul a n= z 2 (p)(1-p) / (er ror 2 ), wher e n is sampl e siz e, z is standard -sco re at 95% confidenc e int erval =1.96, p is the prevale nce of bre ast canc er, and error = 5% (Significant diffe renc e). Using a prevalen ce rat e of 4.03% (esti mated) obt ained from the 2009 annual repo rt from the Depa rtment of Pathol og y, UGMS , Korl e - B u in Ghana, a minim um of 59 breast cancer pati ents w ere need e d for thi s stud y. 3.5 Informed con sen t. T he study was gi ve n approva l by the Ethi ca l and Prot oc ol Revi e w Comm it t ee of the Unive rsi t y of Gha na Medi c al School , Coll e ge of Hea lt h Scie nc e , Korl e - Bu. The prot oc ol ide nti fi c a ti on numbe r of the ethic al clea ranc e for this wor k was MS-E t / M.1-p 5.1/ 2011 -12 (Appendi x III). The refe re nc e number was MS-A A/ C.2/Vol.16A. Conse nt was sought from the Hea d of De pa rt me nt of Pathol ogy, Unive rsi t y of Medi c al School for the use of arc hi ve d patients¶ form al i n -fi xe d para ffi n embe dde d brea st tissue bloc ks. 3.6.0 Use of con trol sa mp les. 3.6.1 Positive con trols For Oestrog en Recepto r and Prog este ron e Receptor Archived fo rmali n fix ed paraf fin wax embedded t iss ue blocks of a hist ologi call y diagnose d fibroad enoma of a 12 yea r old femal e. University of Ghana http://ugspace.ug.edu.gh 30 For Human Epider mal Growth Facto r Recepto r -2: Archived fo rmali n fix ed paraf fin wax embedded t iss ue blocks of a hist ologi call y diagnose d invasive breas t cance r tiss ue. 3.6.2 Negative con trols. For ER, PR and HER -2/ neu: Secti ons from same sampl es used as posi ti ve controls wit h the omis sion of the primar y anti bod y incub ati on step. 3.7.0 Sample prep aratio n. S elected formalin - fix ed, paraf fin wax embedde d tissue blocks of pat ient and controls were re- embedded and trimm ed to ex pose the full surfac e of the emb edded tissues. Blocks were put on i ce for 30 minutes to all o w the embedd ed tissues as well as the paraf fin wax att ains an equal cool temperatur e. Using the microtome at a predete rmined gau ge of 4 microns, s ecti ons were cut and all ow e d to float on water bath at a temperatur e of 60 0 C to spread the fo lds in the se cti ons. Secti ons wer e pick ed onto mi croscope sli des and label ed with pati e nt hist opathol og y numb er and arran ged int o s taining racks. Subsequentl y, secti ons were kept in the oven at 60 0 C and then stained with haematox yli n and eosin (H&E) for confirmation of dia gno sis by th e prin cipal su pervisor. After confirmation, one se cti on ea ch fo r ER, PR and HER -2/neu as well as cont rols wer e tak en and kept in staini ng rac ks in the oven at 32 0 C overnight to ensu re prop e r adher enc e of secti ons onto pre-co ated sli des . Comm erciall y pr epared pre- coated sli des obtained from Lei ca Micros ystems wer e us ed. University of Ghana http://ugspace.ug.edu.gh 31 3.7.1 Haematoxyli n &E osin stain in g tech n iq u e. S ecti ons were remov ed from the oven and de wax ed in 3 ch an ges of x ylen e for 2 minutes each. Se cti ons wer e take n through desc ending gra des (80%, 70 % and 60 % ) of eth anol for 2 minutes each. Subsequentl y, sec ti ons wer e placed unde r running tap water for 10 minutes. Nuclear staini ng was done b y incubati n g s ecti ons in aqueous hae matox yli n for 5 minutes with subseq uent blui ng at pH 6.8. Eosin was used for a minute as a count erstain to stain the cytopl as m of the cell in the secti on. Deh ydrati on throu gh asc ending grad es (60%, 70% and 80% ) of ethanol to absol ute ethanol for 2 minutes each was done. Secti ons were subs eque ntl y cle ared in 2 chan ges for 2 minutes each in x ylene and mount ed in Distrene, Pla sti ciser, X yl e ne (DP X) m ountant and cove rslip p ed appropriatel y. Ex ami nati on of the stain ed sli des b y th e Principa l Supervisor was done under the light microscope usin g the X10 as well as the X 40 objecti ves of Ol ym pus microscope, CX41RF. X10 obje cti ve was fo r sc anning th rou gh the secti on and X40 objecti ve fo r detailed evaluation to confirm diagnosis of patients¶ cases. 3.7.2 Immunoh istoch e mist ry. This is based on the prin cipl e that anti bodies bind specificall y to anti gens t hat induced the producti on of the anti bo dies. Th e method provid es a valuabl e means of visuali z ati on of anti gens on froz en se cti ons as well as para ffin wax embedded se cti ons. Th e int roducti on and use of pol ymeric lab eli ng has led to inc re ased sensiti vit y of th e IHC m ethod. University of Ghana http://ugspace.ug.edu.gh 32 3.7.3 Immunoh istoch e mical staini n g proced u r e. Prior to undertaking thi s procedur e, the dil uti ons of the prim ar y anti bodies were vali date d on a series of known posit ive and negati ve contro ls . All staini ng procedure s were car ried out at 25 o C . 3.7.4 Hydration of sections. S ecti ons of each cas e fo r ER, PR and HER -2/neu as well as the posi ti ve and ne gati ve control sli des, which ha ve be en kept in the ove n at 32 o C were t ransf err ed int o a 60 o C oven for 30 minutes. The secti ons were remov ed from the oven, dewax ed in 2 changes of x ylen e for 2 minutes ea ch. Secti ons wer e tak en through 80 % alcohol, 70 % alcohol and 60% alcohol for 2 minute s each and finall y to disti ll ed water. 3.7.5 Epitope retrieval. This is achieved throu gh the breakin g of pr otein cross -li nks formed b y fo rmali n fix ati on thereb y uncov erin g hidden anti genic sit es . The he at induced anti gen ret riev al method was used. A p yr ex bowl wa s filled with 500ml of 0. 01M cit ric acid at PH 6.0 (retriev al solut ion), covered with its lid and then immersed int o a metallic bowl filled with wate r. Enough wate r was used to ensur e that its lev el was at the same l evel as the retriev al solut ion in the pyrex bowl. A hot plate was used to serve as a sour ce of heat . The retri eval solut ion was heat ed to a temperatur e of 95 o C wit hout boil ing. The lid of the p yrex bowl was ca refull y removed and with the help of a hol der, the h yd rated sli des i n its rack was care full y immersed int o the heated retriev al solut ion. The lid of the pyrex bowl was replac ed and se cti ons were all owed to heat for 2 minutes. The p yre x bowl was University of Ghana http://ugspace.ug.edu.gh 33 subsequentl y remov ed from the water bath and allowed to cool to room temperature, after which the slides wer e re moved and plac ed in a bowl of dist il led water. 3.7.6 Incub ation with antibod ies and other rea gen ts. S ecti ons were rinsed in 50m M Tri s-buffer ed sali ne (TBS ) at PH 7.6, and arran ged onto staini ng racks, aft er whic h endogenous pe rox idase acti vit y from, for ex ampl e blood in the tissue, was blocked usin g 3% h ydro gen perox ide solut ion for 5 minutes. Secti ons wer e rinsed in TBS aft er whic h an y protein, ap art from ER, PR and HER -2/neu were blo cked with a protein block solu ti on provided for 5 minu tes. Secti ons wer e then rinsed in TBS . Secti ons were incubate d with 100 ul of primar y anti bodies at the pre-dete rmined concentr ati ons (App endix I) for 60 minutes. The staini ng racks were kep t moi stened to provide enou gh moi stur e that did not all ow th e secti ons to dr y. Afte r the prim a r y anti bod y incubati on per iod, the sli des were rinsed in TBS for 5 minutes and then incubated with 100 ul of the post prim a r y blo ck provided for 30 minutes. Foll owing the rinsing of the sli des in TBS for 5 minutes, slides were incubat ed with 100 ul of Novo Link Pol ym er provided for 30 minutes. Sli des were washed in TBS for 5 minutes with gentl e rocking. Perox idase acti v it y was developed with Diami nobenz idi ne (DAB) for 5 minutes. The DAB rea cti on was s topped with dist il led water afte r which sli des wer e rinsed under running tap water. Secti ons wer e then counte rstained in Haematox yli n, washed und er running t ap wat er, deh ydrated in alcohols of in creasin g grad es, cle ared in x ylen e an d mount ed in Dist rene, P lasticiser, Xylen e (DP X). After the mount ant had been all owed to set, the sli des were ex amined under the light microscope ( Ol ympus micros cope CX41RF) at X10 . The sli des were subsequ entl y revi ewed by th e Pathol ogist . The stained mount ed University of Ghana http://ugspace.ug.edu.gh 34 sli des were confi rmed by review ers usin g th e Ol ympus mic roscope CX41RF. X10 objecti ve was used to scan throu gh the se cti ons and X40 objecti ves use d for detailed evaluation of ER, PR and HER -2/neu posit ivi t y. 3.8 Staini n g reaction and rep orting crite ria for immu n ostain s . Breast tum ours were def ined as posi ti ve fo r ER or PR if ther e was •1 tumour nuclea r staini ng in the pres enc e of ex pected rea cti vit y in int ernal and ex ternal con trols. Absence of tum our nucle ar staini n g or pres enc e of < 1% tu mour nuclea r staini ng in the presen ce of ex pected reacti vit y in internal and ex ternal controls was consi dered negati ve. This reportin g criterion is in accordan ce with recomm e ndati ons made b y th e American Soci et y of Cli nical Oncolo g y/C o ll ege of Americ an Pathol ogist s (AS C O/CAP ). (Ha mm ond et al ., 2010). HER-2/neu was consi de red posi ti ve if mor e tha n 30% of tum our cell s showed stron g compl ete membranous s taining. Weak, incompl ete membran e staini ng or c ytopl asmi c staini ng was consi dered negati ve. All the immunos tained sli des wer e review ed by th e Pathol o gist , afte r the principal investi gator had repo rted /scored them. University of Ghana http://ugspace.ug.edu.gh 35 3.9 Statistical analyses. Data obtain ed were ente r ed int o and anal ys ed b y t he Statist ical Pro gramm e for th e Social Sciences (SP S S ) Version 16.0. Fr equen cies, per ce ntages, means and stand ard deviations were used to summ a riz e data coll ected. The pai red sampl e t -test was used to test fo r significant dif fer ences in means. The McN e mar test was used to determi ne an y statis ti call y signifi cant differen ce s in ER, PR and HER -2/neu betwee n prim ar y and correspondi n g l ymph nodes. A probabil it y value less than 0.05 was consi der ed statis ti call y signific ant. University of Ghana http://ugspace.ug.edu.gh 36 CHAPTER 4: RESULTS 4.0 Total numb er of cases. The hist opathol og y lo g book was us ed to retriev e pati ent information su ch as age and the t ype of specim en. In form ati on with regards to diagnosi s, tum our grad e, number of l ymph nodes invol ved with metastatic bre ast tum our wer e retriev ed from the dupl icate repo rts of patients¶ results kept at the department. The total number of surgical cases received at the Department of Pathol o gy in the yea r 2009 was 7,115. About 11% (786 out of 7,115) of these cas es wer e bre ast cases . Out of the tot al nu mber of br east cas es s ub mi tt ed, 36.5 % (287 out of 786) were po sit ive for breast canc er. The rest of the bre ast cas es wer e most l y fibroadenomas. About 40% (114 out of 2 87 ) of the bre ast canc er spe cim e ns were tru cut biops ies and 39% (112 out of 287) were breast can cer cas es witho ut l ymph node metastase s . About 21% (61 of 287 ) of the breast cance r cases wer e mastectom y specim ens with ax il lar y contents, and were dia gnosed as invasive du ct al car cinom as (IDC) with l ymph node metastase s. About 11 % (7 out of 61) of the breast cancer c ases with ax il lar y l ymph nod e metastases were ex cluded bec ause the prim a r y breast cance r tissue showed ex tensive necrosis . The tot al number of cas es that were rec ruited int o the stud y was ther efor e 54. These pati ents rec eived neo adjuvant chemot her ap y as p art of their treatm ent and/ or m ana gement plan. University of Ghana http://ugspace.ug.edu.gh 37 4.1 Pathologi cal chara c teristics of cases. The youn gest pati ent enroll ed in this stud y was 25 yea rs and the oldest was 72 years. The mean age ± SD was 49.8 9 ± 10.70. The median age was 51 years. About 3 0% (16 out of 54) of the cas es were between the ages of 49 and 56 (Table not sho wn). About 20.4 % (11 out of 54) of the pati ents had grad e I br east canc er , 42.6% (23 out of 54) had grade II and 37% (20 out of 54 ) had grad e III breast can c er respe cti vel y. Al l of the cases wer e invasive ductal car cinom as. The number of ax il lary l ymph nod es that were retrieved from the ax il lar y contents ran ged from 1 to 13. The percent a ge of retriev ed ax il lar y l ymph nodes that were invol ved with breast cancer cell s ran ged from 8% to 100%. About 22% (12 out of 54 ) of th e cas es had 100 % l ymph nod es invol ved w it h br east cancer cell s and 3.7% (2 out of 54) had 8% of retriev ed l ymph nodes invol ved with brea st cancer cell s. The mean pe rc enta ge of lym ph nodes that were in volved wit h b reast cance r was 57.87 %. University of Ghana http://ugspace.ug.edu.gh 38 Table 1 shows the sum mar y of ER, PR and HE R -2/neu statuses betw ee n prim ar y breast tum our and corr espondi ng l ymph nodes. Table 1 : Summary of ER, PR and HER-2/neu statu ses in pri ma ry tu mou r and lymp h nod es. Recepto r statu s Primary tu mou r Corresp on d in g Lymph nod e Frequ enc y Percenta ge (% ) Frequ enc y Percenta ge (% ) ER posit ive 17 31.5 18 33.3 ER negati ve 37 68.5 36 66.7 PR posi ti ve 14 25.9 16 29.6 PR negati ve 40 74.1 38 70.4 HER - 2 posi ti ve 14 25.9 16 29.6 HER - 2 ne gati v e 40 74.1 38 70.4 n =54 There was a sli ght incr ease in rec eptor posi ti vit y as well as an incre a se in receptor negati vit y in the l ymph node s (Table 1). Howev er, there was no statis ti cal differ ence, p >0.05, betwe en them. University of Ghana http://ugspace.ug.edu.gh 39 Figu re 3 shows micro gra phs of control slides. A B C D Figu re 3: Sli des of a fib roadenoma tissue showi ng; A: negati ve con trol , B: PR posi ti ve control, C: ER posi ti ve control and D: HER -2 /ne u posi ti ve control in an i nvasive ductal carcinom a tissue (X400 for all sli des ). University of Ghana http://ugspace.ug.edu.gh 40 Figu res 4 A and B sho w micrographs of ER posi ti ve sli des of a primar y breast tum our and a l ymph nod e resp ect ivel y. Figure 4 A: ER positive prima ry tu mou r (X400). B: ER positive lymp h n node(X400). Figu res 5 A and B show microgr aphs of P R positi ve sli des of a prim ar y breast tum our and l ymph node respe cti vel y. Figure 5 A: PR positive pri mary tumou r (X400) B: PR positive lymp h nod e (X400) University of Ghana http://ugspace.ug.edu.gh 41 Figu res 6 A and B show micrographs of HER -2/ neu posi ti ve sli des of a prim ar y bre ast tum our and l ymph node respecti vel y. Figure 6 A: HER-2 positive primary tu mou r(X400). B: HER-2 positive lymp h node(X400). Case to case comparison of ER, PR and HER -2/neu between prim ar y br e ast cance r and correspondi n g l ymph no des (Tabl e not shown ) s howed that, o f the 54 cases an al ys ed, 1. 9% (1 out of 54) was posi ti ve for ER in the prim ar y br east tum our and negati ve in the l ymph nodes. About 4% (2 out of 54 ) of th e cases wer e ne gati ve fo r ER in the prim ar y breast tum our and posi ti ve in the l ymph nodes. The discordant rate fo r E R was 5.6%. There was 94.4% (51 ou t of 54) concordan ce fo r ER between prim ar y br e ast canc er and metastatic deposi ts in l ymph nodes. The discordant rate for PR was 7.5%. About 2 % ( 1 out of 54) of the cases that were PR posi ti ve in the prim ar y tum our was negati ve in the correspondi n g l ymph no de metastasis . About 6% (3 out of 54) of the cas es which were PR negati ve in the prim a r y tum our were posi ti ve i n the co rrespondi n g meta static deposi ts University of Ghana http://ugspace.ug.edu.gh 42 in the l ymph nod es. There was 92.6% (50 out of 54) concord anc e fo r PR between prim ar y breast can cer an d metastatic deposi ts in lym ph nodes. None of the HER-2/neu posi ti ve prim ar y br east tum our was negati ve in the metastatic deposi ts in the correspondi n g l ymph no des. About 4% (2 out of 54) of the HER -2/neu ne gati ve prim a r y tum or were posit ive in the corr espondi ng ax il lar y l ymph nodes. The re was 96.3% (52 out of 54) conco rdanc e for HER -2/neu. Table 2 shows the subt yp es of bre ast can cer as det ermined b y ER, PR and HER -2/neu statuses. Table 2: Subtypes of breast can ce r ac cord in g to rec ep to r statu s. Subtype Frequen cy Percen tage (% ) Tripl e ne gati ve (ER - /P R - /HER - 2/neu - ) 26 48.1 HER - 2/neu typ e (ER - /P R - /HER - 2/neu+) 6 11.1 Lumi nal A (ER +/P R + - /HER - 2/neu - ) 12 22.2 Lumi nal B (ER+/P R + - /H ER - 2/neu+) 5 9.3 ER - /P R +/HER - 2/neu+/ - 5 9.3 Total 54 100 Triple ne gati ve br east ca nce r was the most frequ e nt followed by the Lumi nal A typ e and then the HER-2 t yp e br ea st cance rs. Hormone rec e ptor posit ive and HER -2/neu posit ive breast cance r (ER+/P R +/HER2+), as well as ER negati ve breast can cers were infrequ ent. University of Ghana http://ugspace.ug.edu.gh 43 4.2 Pathologi cal chara c teristics amon g trip le negative breas t can ce r ca ses. About 48% (26 out of 54) of the cases that wer e recruited in thi s stud y had tripl e negati ve breast cance r (T NBC ). T he age ran ge amon g thi s subt ype of bre ast can ce r was 29 to 7 2 yea rs. The mean age ± SD was 49.89 ± 10.77. The median age of the pati ents with TNBC was 50 years . About 15 % (4 out of 26) had gr ad e I breast canc er, 50 % (1 3 out of 26) and 34.6% (9 out of 26) had gr ade II and grad e III bre ast canc er resp ecti vel y. The perc enta ge of l ymph node invol vement among thi s group of breast can cer was 8% to 100%. About 39% (10 out of 26 ) pati e nts had 100% l ymph nod es invo lved wit h breast cancer cell s. There was no statis ti cal differenc e in age (p=0.853), tum our grad e (p=0.731) and percent a ge of l ymph nod es invol ved with metasta ti c breast cance r (p =0.32 5) betwe en the TNBC sub group and th e other groups of breast ca ncer. University of Ghana http://ugspace.ug.edu.gh 44 Table 3 shows the com parison of the differ enc es in ER, PR and HER-2/neu betwe en prim ar y breast can cer an d correspondi n g l ymph n odes. Table 3 : Comparison of dif f eren ces in ER, PR and HER-2/neu betw een prima ry breast can ce r and corr e sp on d in g lymp h nod es. Recepto rs Primary tu mou r Corresp on d in g lymp h nod e P valu e ER Posit ive 17 (31.5) 18 (33.3) 1.000 Negati ve 37 (68.5) 36 (66.7) PR Posit ive 14 (25.9 16 (29.6) 0.625 Negati ve 40 (74.1) 38 (70.4) HER-2/neu Posit ive 14 (25.9 16 (29.6) 0.500 Negati ve 40 (74.1) 38 (70.4) The McN emer t est was u sed to test for an y si gnifi cant diff eren ces i n the ex pressi on of ER, PR and the over ex pression of HER -2/neu between the prim ar y bre ast cancer and the correspondi n g metastatic deposi ts in the axil lar y l ym ph nodes . Ther e were no statis ti call y significant diff eren ces (p >0.05) in ER, PR and HER -2/neu between prim a r y br east can ce r and corr espondi n g l ymph nodes (Table 3 ) . University of Ghana http://ugspace.ug.edu.gh 45 CHAPTER 5: DISCUSSION The incidenc e of bre ast canc er va ries globall y. Esti mated worldwide inci dence o f br east canc er ran ges from 16.8 per 100,000 wom en in West Africa to 89.7 per 100,00 0 women in Western Europe (G LOBOCAN 2008). In Africa, the esti mated incidence of bre ast canc er ran ges from 16.8 per 100,000 in Guinea to 41 per 100,000 in South Africa. The esti mated br east can cer incide nce in Gh ana as reported in the GLO B OCAN Cance r Fact Sheet is 25.8 per 100,000 . However, the stud y pres ented herein sho wed that out of the 786 breast cases, 287 (36.5% ) were hist olo gica ll y dia gnosed as breast ca ncer. Breast canc er occurs at a relativel y yo un ger age i n Afri can women as com pared to Whit e wo men. The mean age of breast can ce r at dia gno sis among Afric an wom en is 48 yea rs (Rambau et al ., 2011). Sim il arl y, in thi s stud y, th e mean age of br east canc er was 49.87 ± 10.70 ye ars, which is con sis tent with findings in th e African medical liter at ure. In Europ e (Whit es), the median age at diagnosi s of breast ca ncer is reported to be 67 yea rs ( Finni sh Cancer Re gist r y, Canc er stat fact sheets , 2011). The median age at dia gnosi s of breast canc er as reported in thi s stud y was 51 years. Th e factors responsi ble for thi s difference in age at dia gnosi s of br east can cer ar e not full y understood, alt hou gh it could be due to mut ati ons in the bre ast can cer suscepti bil it y genes BRCA 1 and BR CA 2 and th eir variants which are mor e comm on among Bla c k (Afric an) women (A meri can Canc er Societ y, 2011 -2012 ). About 80% of the cases accesse d in thi s stud y were gr ade II and III br east canc ers . In addit ion, the av era ge l ym ph node invol vem ent b y metastatic br east canc er was 60%. Thi s is consi stent with report s t hat Afric an women present late to hospi tals with advanc ed University of Ghana http://ugspace.ug.edu.gh 46 breast canc er due to late att endan ce or reportin g to the hospi tal as repo rte d by Harris et al ., (2000) and Cle gg- La mpt e y et al ., (2007 ). In thi s stud y, the ex pressi on of ER was 32 % an d is sim il ar to ER ex pressi on of 23% in African women as repor ted by Olopade (2005 ). The ex pr essi on of ER in breast cance r among wom en in the Uni ted States is 70 -80% (Go wn, 2008). The low er pre valence of ER ex pressi on in Afric an wo men as compared to ER ex pressi on in Whit es, coupled with the high preval ence (48.1 %) of tripl e negati ve bre ast cancer in Afric an women as observed in thi s stud y, tends to make breast can ce r in African women more aggr essi ve (Rambau et al ., 2011) than bre ast canc e r in Whit es. The preval ence of t riple ne gati ve breast c ance r worldwide is 15% (Lar a -Medina, 2011). The rea son(s) for the hi gh preva lence of tripl e negati ve br east canc er co uld not be disc erned fro m the stud y, alt hou gh ac cording to Stark et al ., (2010), geneti cs m a y pla y a role. Fu rther st udies need to be done to define the hi gh prevalen ce of t riple ne gati ve breast canc er among Gh anaian and other African women with breast can cer. 5.0 Comparison of ER, PR and HER-2/neu. Bre ast canc er is consi dered as a hetero geneous disease (Perou et al ., 2000). As breast can c er pro gr esses, clona l ex pansion of breast cancer cell s occur and t hese cell s ma y become geneti c all y alt e re d leading to tum our heter ogen eit y (Almendro and Fuster, 2011). These geneti c alt erati o ns ma y contribut e to the diffe renc es in ho r mone receptor ex pres sion and HER-2/neu over ex pressi on betwe en prim ar y bre ast canc er and metastatic deposi ts in correspondi ng l ymph nod es as obser ved in thi s stud y. Durin g br east can ce r University of Ghana http://ugspace.ug.edu.gh 47 progressi on, there is also the adaptation of clones of tum our cell s in chan ging microenvironments. In th e l ymph node mic roenvir onment, accordin g to Dawood (2000) clonal selecti on of tum our cell sub populations occurs and thi s could acco unt for some of the metastatic deposi ts in the l ymph nodes bein g hormone recepto r ne gati ve whiles the prim ar y tum our is hormone rec eptor posit ive and vice vers a. Lit er ature has rev ealed var yin g result s from th e comparison of ER, PR an d HER -2/neu between primar y breast canc er and metast ati c tum our deposi ts. Fact ors that ma y contribut e to differenc es in the ex pres sion of ER, PR and the over ex pressi on of HER - 2/neu betwe en prima r y breast canc er and metast ati c tum our deposi ts ma y i nclude: tum ou r hetero gen eit y (Nede r ga a rd et al ., 1995; Azam et al ., 2009; Arslan et al ., 2011), clonal selecti on of tum o u r cell subpopul at ions , geneti c inst abil it y of tum o u r cell s (Edge rton et al ., 2003; Prat and Perou, 2009) , local or systemi c treatm ents, the time interval betwe en prim ar y tum o u r and metastasis , recepto r status determi nati on techniques (Prat and Perou, 2009), and the site of met asta sis (Arslan et al ., 201 1). S im mons et al ., 2009 and Broom et al ., 2009 reported in their studi es that there was no significant con cordan ce for ER, PR and HER -2/neu betw een prim ar y br e ast tum our and dist ant metastases . Contr ar y to the findin gs of Sim mons et al ., (20009) an d Broom et al ., (2009), the findings rep orted herein indi cat e a significant con cord ance i n ER, PR and HER-2/neu between primar y breast tum our an d metastatic deposi ts in l ymph nod es. Sim il ar findi ngs wer e rep orted by Holdaw a y et al ., (1983 ) and Azam et al ., (2009 ). University of Ghana http://ugspace.ug.edu.gh 48 5.1 Conclu sion. There are minor chan ges between ER, PR and HER -2/neu status between primar y tum our and metastatic deposi ts in l ymph nod es. Thes e chan ges are how eve r, statis ti call y not significant . In the absenc e of the prim ar y br east cancer tissue, the tum our deposi ts in the resect ed ax il lar y l ymph nodes will give a fair re flecti on of the ER, PR and HER -2/neu status of the prim ar y tu mour in the majorit y of cases to inform further management decisi on. 5.2 Recommen d ation. In the absenc e of the pr im ar y bre ast cance r tissue, the tum our deposi ts in the res ected ax il lar y l ymph nodes will give a fair reflecti on of the ER, PR and HER -2/neu status of the prim ar y tum our in the majorit y of cas es to inform further man a gement decisi on. University of Ghana http://ugspace.ug.edu.gh 49 REFERENCES Adamcz yk A., Niemi ec J ., Ambicka A ., Maáecki .., W ysocki W.H ., MituĞ J. and RyĞ J. (2012). Ex pressi on of ER/P R /HER2, basal markers and adhesion mol e cu les in prim ar y breast canc er and in l ym ph nodes metastases : a compa r ati ve immun ohist ochemi cal anal ysis . Pol J Pathol , 63 (4):228 -234. Ad isa C.A ., Elew eke N ., Alfred A.A ., C ampbell M.J ., Sharm a R ., Nse yo O ., et al . (2012). Biol og y of brea st canc er in Nigerian women: a pil ot stud y. Ann Afr Med . , 11(3):169 -75. Aktas B ., Müller V ., Tewes M ., Zeit z J ., Kasim ir-B auer S ., Loehb er g C.R ., et al . (2011). Comparison of estro gen and pro gesteron e recept or status of circulatin g t umor cell s and the prim ar y tum or in metastati c breast canc er pati ents. Gynecol Oncol , 122 (2): 356 -360 . Almendro V . and Fuster G . (2011). Het ero gen eit y of breast canc er: eti olo gy and cli nical relevan ce. Clin Transl Oncol ., 13(11):767 -7 73 . Alro y I. and Yar den Y. (1997). The ErbB sign all ing n etwork in embryo gen esis and oncogenesis : signal div ersificati on throu gh co mbi natorial ligand -r ecep tor int eracti ons. FEBS Lett, 410 : 83-86. American Canc er Societ y Breast Canc er Fa ct and Figur es, 2011 -2012. ww w.goo gle. com. Access ed 17/2/ 12. University of Ghana http://ugspace.ug.edu.gh 50 Anderson J .N., Peck E .J. and Clark J .H. (1975). Estrogen -induced ute rine responses and gro wth: Relations hip to recepto r estro gen bindi n g b y uterin e nucl ei. Endocrinology 96 : 160. Andre F ., Sli mane K ., Bach elot T ., Dunant A ., Namer M ., Ba rreli e r A . , et al . (2004). Bre ast can cer with s ynch ronous metastases: trends in survival during a 14 - year period. J Clin Oncol , 22(16): 3302 -3308. Ng. A. K. and Travis L.B. (2009). Radiation Therap y and Br east Canc er Risk. J Natl Compr Canr Netw , 7: 1121 -1128. Andruli s I. L., Bull S. B. , Bla ckstein M.E., Suth erland D., Mak C. , Sidl ofsk y S. , et al . (1998). neu / Erb B - 2 ampl ificati on identifies a poor - pro gnosi s group of wom en with node - negati ve breast can cer. J Clin Oncol, 16 : 1340 ±1349. Antoniou A.C., Shenton A., Maher E.R., Watson E., Woodward E., La ll oo F., et al . (2006). Parit y and bre a st cancer risk among BRCA1 and BRCA2 mutation carriers. Breast Cancer Research , 8 : R72. Archampon g E.Q. (1977) . Breast Cance r. Ghana Med J, 16 (2 ): 63. University of Ghana http://ugspace.ug.edu.gh 51 Arslan C ., Sa ri E ., Akso y S . and Altunda g K . (2 011). Variati on in hormo ne receptor and HER-2 status between pr im ar y and metastatic br e ast canc er : revi ew of the literature. Expert Opin Ther Targets , 5(1): 21 -30. Azam M., Qureshi A. and Mansoor A. (2009). Comparison of Estrogen recepto rs, Progeste rone receptors and HER -2/neu Ex pressi on between Primar y and Metastati c Breast Carcinom a. JPMA , 59 : 736. Badoe E.A., Baak o B.N. The Breast. Bako e E.A., Acheampon g E.Q. and da Rocha -Afodu (edit orials). (2000). Princ ipl es and Practi ce of Sur ge r y Includi n g Pathol og y in the Tropics, Accr a: Depa rtment of Surger y, Unive rsit y of Gha na Medical School, 449 - 477. Barn es D. M. and Hanb y A. M. (2001). Estro ge n and Progesteron e rece ptors in breast canc er: past, pres ent and future. Histopathology, 38 : 271 -27 4. Barton S . and Swanton C . (2011). Recent devel opments in treatm ent strati ficati on for metastatic breast canc er. Drugs, 71(1 6): 2099 -211 3. Baue r K.R ., Bro wn M ., Cress R.D ., Parise C.A . and Caggiano V . (200 7). Descriptiv e anal ysis of estro gen rec eptor ( ER ) -ne gati ve, pro geste rone receptor ( PR )-ne gati ve, and HER2-ne gati ve invasiv e breast can cer, the s o-call ed tripl e -ne gati ve phenot ype: a population -based stud y from the Cali fornia can cer Regist r y. Cancer , 109 (9) : 1721 -172 8. University of Ghana http://ugspace.ug.edu.gh 52 Baue r K., Parise C . and Caggiano V . (2010 ). Use of ER / P R / HER2 subt yp es in conjunction with the 200 7 St Gall en Consensus Statement for earl y breast canc er . BMC Cancer , 10: 228. Beckh ardt R.N., Ki yoka wa N. and Xi L. (1995) . HER - 2/neu onco gen e ch ar act eriz ati on in head and neck squ amou s cell car cinom a . Arch Otolar yn gol Head Neck Surg, 121(11 ): 1265- 1270 . Benöhr P ., Henkel V ., Speer R . and Vogel U . (2005). Her - 2/neu ex pre ssi on in breast canc er -- A compa rison of differ ent dia gnost ic methods. Anticancer Res . , 25(3B): 1895 - 1 900. Ber r y D.A., Cronin K.A., Plevrit is S.K., Fr yback D .G., Clarke L., Zelen M ., et al . (2005). Effect of S cre ening and Adjuvant Ther ap y on Mortalit y from Br east Can cer. N Engl J Med, 353: 1784 -1792. Biritwum R.B., Gulaid J. and Amaning A. O. (2000). Patt ern of diseases or condit ions leading to hospit ali sati o n at the Korle Bu Teachin g hospi tal. Ghana Med J, 34 : 197 ±205. Bo gina G., Bort esi L . , Marconi M ., Venturini M ., Lun ardi G ., Coati F ., et al . (2011). C omparison of hormonal rec eptor and HER -2 stat us between br east prim a r y tum ours and relapsin g tum ours: cli ni cal impli cati ons of pro gesterone receptor loss . Virchows Arch, 459(1): 1 -10. University of Ghana http://ugspace.ug.edu.gh 53 Broom R.J ., Tang P.A ., Sim mons C ., Bordeleau L ., Mulli gan A.M ., O'M all e y F.P ., et al . (2009). Changes in estrogen recepto r, progester one recepto r and Her -2/ neu status with time: discordance rates between prim ar y and m etastati c breast can cer. Anticancer Res, 29(5): 1557 -1562. Brown M ., Tsodikov A ., Bau er K.R ., Pa rise C.A . and Ca ggiano V . (200 8). The rol e of human epiderm al growt h facto r recepto r 2 in the survival of wom en wit h estro gen and progesterone recepto r -ne gati ve, invasive bre ast cancer : the Cali fo rnia Ca ncer Re gist r y, 1999-2004. Cancer, 112 ( 4): 737 -747. Campagnoli C., Clav el -C hapelon F., Kaaks R., Peris C. and Berr ino F. (2005). Progesti ns and pro gester one in hormone replac ement therap y and the risk o f breast canc er . J Steroid Biochem Mol Biol, 96 : 95 ±108. Citri A . and Yarden Y . (2006). EGF -ERB B si gn all ing: towards the s yste ms level. Nat Rev Mol Cell Biol . , 7(7): 505 -516. Clark G. Prognost ic and predictive fac tors. In: Harris J R. (2000). edit or. Diseases of the breast, Phil adelphi a: Lip pincott Wil liams & Wil kins, 2: 489 ±514. Clarke M. (2006). Meta -anal ys es of adjuvant therapies for women wit h earl y br east cancer the Early Breast Cancer Trialists¶ Collaborative Group ove rview. Ann Oncol, 17 (10): 59 ±62. University of Ghana http://ugspace.ug.edu.gh 54 Clegg- Lampt e y J .N.A. and Hodasi W.M. (2007). A Stud y of Breast Canc er in Korle Bu Teachin g Hospit al: Asse ssi ng the Impact of Healt h Educati on . Ghana Med J., 41 (2 ): 72 ± 77. Coldi tz G.A., Wil lett W.C. and Hunte r D .J . (1993 ). Famil y hist or y, age, an d risk of breast canc er. Prospecti ve data from the Nurses ' Healt h Stud y, JAMA, 270 (3): 3 38 - 343. Condon J .C., Hard y D. B., Kovaric K. and Mend elson C.R. (2006). Up -r egul ati on of the pro gesteron e rece ptor (PR ) -C isoform in laboring m yometrium b y acti vati on of nucle ar factor -kapp a B ma y contribut e to th e onset of l abor throu gh inh ibi ti on of PR functi on. Mol Endocrinol , 20: 764 -775. Cork D. , M. W., Lenna r d J .T.W . and Tyson -C ap per A.J . (2008). Alternati ve spli cing and the progesteron e re ceptor in breast can cer. Breast Cancer Research , 10(3): 207. Cowherd S.M . (2012). Tumor stagin g and gradin g: a prim e r. Methods Mol Biol., 823 : 1 - 18. Cui X. , Schiff R. , Arpi no G. , Osborn e C. K. and Lee A.V. (2005 ). Biol og y of progesterone re ceptor los s in breast can cer and its impli cati ons for endocrin e therap y. J Clin Oncol , 23: 7721 - 7 735. Dalvai M. and Bystri ck y K. (2010). Cell C ycle an d Anti - Estrogen Ef fects S yner giz e to Regulate Cell Prolifer ati on and ER Tar get Gen e Ex pressi on. PLoS ONE , 5(6). University of Ghana http://ugspace.ug.edu.gh 55 Dawood S . (2010 ). Tripl e -ne gati ve bre ast cance r : epidemiol og y and man a gem ent opti ons. Drugs, 70 (17): 2247 -22 5 8. Dawood S., Bro gli o K., Buz dar A.U., Hortoba g yi G.N. and Giordano S.H. (2010). Progno sis of Women w i th Metastatic Br east Can cer b y HER2 Status and Trastuz umab Treatm ent: An Insti tut ional - B ased Review. JCO, 28 (1): 92 - 98. Dent R. , Trudeau M. and Pritchard K. I. (2007) . Tripl e - ne gati ve bre ast cancer: cli nical featur es and patt e rns of recurr en c e . Cli n Cancer Res , 13(15 Pt 1 ): 4429 - 44 34 . De Sombre E.R., Mohl a S. and Jenson E.V. (1975). Receptor t ransform ati on, the ke y to estrogen acti on. J Steroid Biochem, 6 : 469 - 473. Dhingr a K. (1999). Anti estrogens --tamox ifen, S ERM s and be yond. Invest New Drugs, 17(3): 285-311 . Duff y M.J ., McGowan P.M . and Crown J . (2012). Tar geted the rap y for tripl e-negati ve breast cance r: wher e ar e we? Int J Cancer , doi: 10.1002/i jc.27632 . Earl y Br east Canc er Tri ali sts ' Co ll aborati ve Gro up. (1998). Tamox ifen for earl y br east canc er: An ove rview of t he randomi sed trials. Lancet , 351: 1451 ±1467. Early Breast Cancer Trialists¶ Collaborative Group. (2005). Effects of chemotherapy and hormonal therap y for ear l y br east ca nce r on recurr ence and 15 - ye ar surviv a l: an overview of the randomi z ed trials. Lancet , 365 : 1687 ±1717. University of Ghana http://ugspace.ug.edu.gh 56 Edge rton S.M. , Moore D. And Merkel D. (2003). E rbB - 2 (HER - 2) an d breast canc er progressi on . Appl Imm u nohis tochem Mol Morphol , 11: 214 - 221. Eltz e E. , Wülfing C. , Von Struensee D. , Piechota H ., et al . (2005). C ox - 2 and Her2/neu co - ex pressi on in invasive bladder cance r . Int J Oncol, 26 (6 ): 1525- 1531 . Enmark E., Pelt o -Huikk o M. and Grandien K.( 1997). Human estro gen rec eptor bet a - gen e structur e, chromos omal locali z at ion, and ex pressi on patt ern. J Clin Endocrinol Metab , 82 : 4258 ±4265. Ferla y J ., Shin H. R., Forman D., Mathers C. and Parkin D.M. (2010). Esti mates of Worldwide Burde n of C ancer in 2008: GLO BOC AN 2008. Int J Cancer , 127 (12): 2893 - 2917. htt p:/ /globocan.air c.fr . Access ed 14/02/ 2012. Finni sh Cancer Re gist r y, Can cer stat fact sheets, Insti tut e for Statist ical and Epidemi ological Canc er Research, Helsi nki, Finl a nd, 2011. Fowler A.M., Santen R. J . and Allred D.C. (200 9). D wa rf estro gen in br east can cer and resis tance to tami x ifen. J Clin Oncol , 27(21): 3413 -3415. Gancbe r g D ., Di Leo A ., Cardoso F ., Rouas G ., Pedrocchi M ., Paesmans M ., et al . (2002). Comparison of HER -2 s tatus between prim a r y breast canc er and corr es p onding dist ant metastatic sit es. Ann Oncol ., 13(7): 1036 -10 43. University of Ghana http://ugspace.ug.edu.gh 57 Gipponi M., Bassetti .C, Canavese G., Catt urich A., Di Somm a C., Vecchio C., et al . (2004) . S enti nel l ymph node as a new mark er for therapeuti c plannin g i n breast can cer pati ents. J Surg Oncol, 85 (3): 102-111. Girett i M.S ., Fu X.D ., De Rosa G ., Sarotto I ., Balda cci C ., Garibaldi S ., et al . (2008). Ex tra-nuclear si gnall ing of estrogen receptor to breast can cer c ytosk elet al remodelli ng, migrati on and invasion. PLoS One. , 3(5): e2238. G LOBOCA N 2008 Can cer Fact She et. Br east Cancer In cidenc e, Mort ali t y and Prevalenc e Worldwide in 2008. www. goo gle.com . Access ed 12/3/ 12. Gluz O., Li edtke C ., Gott schalk N ., Pusz tai L ,, Nitz U . and Harb eck N . (2009). Tripl e-ne gati ve breast cancer --cu rrent status an d future dire cti ons. Ann Oncol. , 20(12):1913 -19 27 . Gorski J . and Raher B. (1974). Estro gen acti on in the uterus: The requisi te for sust aine d estrogen bindi ng in the nucleus. Gynecol Oncol, 2 : 249. Gown A.M. (2008). Current iss ues in ER and HER2 testin g by IHC in bre ast cance r. Mod Pathol , 21: S8 ±S 15. University of Ghana http://ugspace.ug.edu.gh 58 Graham J .D., Yeates C., Ball ein e R. L., Harv e y S.S ., Mill iken J .S , Bil ous A.M. , et al . (1995). Char acteriz ati on of pro geste rone recepto r A and B ex pressi on in human breast canc er. Cancer Res , 55: 5063 - 5068. Gravalos C. and Jim eno A. (2008). HER2 in gast ric canc er: a new pro gno sti c factor and a novel therap euti c tar get. Ann Oncol., 19 (9): 152. Griffths C. L . and Olin J .L . (2012). Triple Negati ve Breast Can ce r : A Brie f Review of its Characte risti cs and Tre at ment Options . J Pharm Pract , Guarne ri V., Giovannell i S., Ficarra G., Bett ell i S., Maiorana A., Piacenti ni F., et al. (2008). Comparison of HER -2 and Hormone R eceptor Ex pressi on i n Primar y Breast Cancers and As ynchron ous Paired M etastases: Impa ct on Pati ent Man agement. The Oncologist , 13(8): 838-8 44. Gutierrez C. and Schiff R. (2011). HER 2: Biol og y, Det ecti on, and Cli nical Impl icati ons. Arch Pathol Lab Med ., 135 (1): 55 ±62. Habash y H.O., Powe D. G., Rakha E.A., Ball G. , Macmi ll an R.D, Gree n A.R., et al. (2010). The pro gnost ic s ignifican ce of PE LP 1 ex pressi on in invasive br ea st cance r with emphasis on the ER - p osit ive lum inal - li ke subt ype . Breast Cancer Research and Treatment , 120(3): 603 - 6 12. University of Ghana http://ugspace.ug.edu.gh 59 Haider W., Arain G.M., Sohu K.M., Naqi S.A. and Younus B.B. (20 01). Estrogen recepto rs as tum our mar kers in malign ant breast dis ease: A ret rospecti ve stud y of 100 cases. Biomedica , 17: 1- 5. Hamm ond C.B and Soo ule M.R . (2004). Endoc rine asp ects of ad enoc ar cinom a of the endometrium . In Scia rra J . J. (ed). G yn aecolo g y and obst etri cs CD -R OM. htt p:/ /www.glowm.com/r esources/ glowm/ cd/pa ge s/v4/ v4co14.htm l . Date last accessed, 29/02/ 2012 . Hamm ond M.E ., Ha yes D.F ., Dowsett M ., Allre d D.C ., Hagert y K. L ., Badve S ., et al . (2010). Ame rican Societ y of Cli nical On colo g y/C oll ege of Ame rica n Pathol ogist s guideline recomm end ati ons for immunohi stochemi cal testin g of estrogen and progesterone receptors i n bre ast can ce r ( un abri dged ve rsion). Arch Pathol Lab Med , 134(7): e48 -72. Harr ell J .C., Dye W.W ., Allred D.C., J edli cka P., Spoelst ra N.S., S artorius C.A. , et al . ( 2006). Estro gen Re ce ptor Posit ive Breast Cancer Met astasis : Altered Hormonal Sensit ivi t y and Tumor Aggr essi veness in Lymph a ti c Vessels and Lymph Nodes. Cancer Res, 66 : 9308. Harris, J .R., Lippm an, M.E., Morrow, M., and Osborne, C.K., Eds. (20 00). Diseases of the Br east 2nd Ed., Phil adelphi a: Lippi ncott Wil lams & Wil kins. University of Ghana http://ugspace.ug.edu.gh 60 Hartmann L.C., In gl e J .N. and Wold L.E. (1994). Pro gnost ic valu e of CerbB2 overex pressi on in ax il lary l ymph node -posi ti ve br east can ce r. Result s from a randomi z ed adjuvant treatm ent proto col. Cancer , 74 : 2956 - 29 63. Hegg R , De Souz a A. Z , Pestana C.B . and C ar doso de Almeida P.C . (1990). Tissu e carcino embr yonic anti ge n in the prognosi s of earl y invasive br east cance r . Breast Cancer Res Treat . , 15(3): 213-6. Heldring N., Pike A. an d Andersson S. (2007). Estrogen receptors: how do the y sign al and what are their ta r gets . Physiological Reviews , 87 (3): 905 ±931. Hetz el D.J ., Wil son T.O., Keen e y G. L., et al . (1992). HER - 2/neu ex pressi on: a major prognost ic factor in endo metrial canc er . G yn ecol Oncol, 47 : 179 - 185 . Hilf R., Feldst ein M. L., Savlov E.D., Gibson S.L. and Sen eca B. (1980 ). The lack of relations hip between estr ogen rec eptor sta tus and response to chemot her ap y. Cancer , 46 (12 Suppl ) : 2797 - 2800. Hoefna gel L.D.C., van de Vijver M.J . , Henk -J an van Sloot en , Wesseling P. , Wesseling J .,W estenend P.J . , et al . (2010). Receptor Convers ion in Breast Cancer M etastases. Breast Cancer Research, 12: R75. Holdawa y I.M . and Bo w dit ch J .V . (1983). Variat ion in receptor status between prim ar y and metastatic bre ast can cer. Cancer , 52(3): 479-4 85. University of Ghana http://ugspace.ug.edu.gh 61 Hopp T.A., Weiss H.L., Hilsenbeck S.G., Cui Y., Allr ed D.C., Horwitz K.B., et al . (2004). Br east can ce r pati ents with progeste ron e rec eptor PR -A-rich tu mors have poorer diseas e-f ree su rvi val rates. Clin Cancer Res , 10: 2751 -2760. Horwitz K.B. and McG uire W. L. (1975). Predi cti ng respons e to endoc rine therap y in human breast canc er: a hyp othesis. Scienc e, 189 : 726 -727. Idirisi n ghe P.K ., Thike A.A ., Cheok P.Y ., Tse G.M ., Lui P.C ., Fook -C hong S ., et al . (2010). Hormone recepto r and c-ERB B2 status in dist ant metastatic and locall y recur rent breast cance r. Pathologic correl ati ons and cli nical significan ce. Am J Clin Pathol . , 133(3): 416 -29 . Jacot W., Pouderoux S., Bibeau F., Le a ha C., Cha teau M.C ., Chapell e A. and Romi eu G. (2011). Hormone rec epto rs and HER -2 changes during bre ast cance r pro gr essi on: cli nical impli cati ons. Bull Cancer , 98 (9): 1059 -1070. Joens uu H., Kell okumpu - Lehti nen P. L. and Bono P. (2006). Adjuvant docetax el or vinorelbi ne with or without trastuz umab for breast cancer. N Engl J Med . , 354 (8): 809 ± 820. Johnson N., Lan caster T. and Full er A. (1995 ). The pr evalenc e of a fami l y hist or y of canc er in gene ral pr acti c e. Fam Pract, 12 (3): 287 -289. University of Ghana http://ugspace.ug.edu.gh 62 Kall el I ., Khabir A ., Bou jelbene N ., Abdenn adher R ., Daoud J ., Frikha M ., et al . (2012). EGFR over ex pressi on relates to tripl e ne gati ve profil e and poo r pro gnosi s in breast canc er pati ents in Tunisi a. Br J Cancer , 106 (6): 1107 -16. Kall ioni emi O.P ., Kall ioniemi A ., Kurisu W ., Thor A ., Chen L.C ., Smi th H.S ., et al. (1992). ER BB2 ampl if icati on in bre ast canc er an al yz ed b y fluores cenc e in sit u hybridi z ati on. Proc Natl Acad Sci U S A. , 89 (12 ): 5321 -5325. Kaptain S, Tan LK and Chen B. (2001). Her ±2/n eu and br east can ce r . Dia gn Mol Pathol , 10: 139 - 152 . Kastner P., Krust A., Tur cott e B., Stropp U., Tor a L., Gronem e yer H., et al . (1990). Two dist inct estrogen - re gulate d promot ers gener ate tra nscript s encodin g the two functi onall y different hum an pro geste rone re ceptor forms A an d B. EMBO J, 9 : 1603 - 16 14. Keen J .C. and Davidso n N.E. (2003). The biol og y of breast carcinom a. Cancer , 97 : 825 ± 33. Kwan M. L. , Kushi L.H., W eltz ien E., Maring B., Kutner S .E., Fult on R .S ., et al . (2009). Epidemi olog y of breast cancer subt ypes in two prospecti ve cohort studi es of breast canc er survivors. Breast Cancer Research , 11: R31. University of Ghana http://ugspace.ug.edu.gh 63 Lacroix M., Toill on R.A. and Lecler cq G. (20 04). Stable µportrait¶ of breast tumors during pro gr essi on: data from biol og y, pathol og y and gen eti cs. Endocr Relat Cancer. 11: 497± 522. Lara-M edina F ., Pérez - S ánchez V ., Sa aved ra- P érez D ., Blak e-C erd a M ., Arce C ., Motol a-Kuba D ., et al ., (2011). Tripl e -ne gati ve breast canc er in Hispani c pati ents: hi gh prevalen ce, poor pro gno sis , and associati on w it h menopausal status, bod y mass index , and parit y. Cancer, 117( 16): 3658 -3669. La w M. L., Kao F.T., Wei Q., Hartz J .A., Greene G. L., Za rucki -S chulz T. , et al . (1987). The progesteron e rec ept or gen e maps to human chromos ome band 1 1q13, the sit e of the mammar y on co gen e int - 2. Proc. Natl. Acad. Sci. U.S.A. , 84 (9): 2877 ±288 1. Levin E.R. and Pietras R.J . (2008). Estrogen receptors outsi de the nucleus in breast canc er. Breast Cancer Res Treat , 108 : 351 ±361. Li Y., Wan g J .P ., Santen R. J ., Kim T.H. , Park H., Fan P et al . (20 10). Estrogen Sti mul ati on of Cell Migrati on Involves Multi ple Signali ng P athwa y Inter acti ons . Cancer -Oncogenes , 151 (11): 51 46. Li X., Huan g J ., Yi P., Bamba ra R.A., Hilf R. and Mu yan M. (2004). Single - chain estrogen rec eptors (ERs) reve al that the ER alpha/ beta hete rodimer emul ates functi ons of the ERalpha dim er in genomi c estro gen si gnali ng pathw a ys. Mol. Cell. Biol, 24 (17): 7681± 7694. University of Ghana http://ugspace.ug.edu.gh 64 Lim J N. W., Hewison J ., Chu C.E and Al -Habsi H. (2011). Facto rs influencin g consul tation to discuss fami l y hist or y of can cer b y as ympt omatic pati ents i n primar y car e. J Community Genet , 2(1) : 19 ±26. Lin N .U ., Vande rplas A ., Hugh es M .E ., Theri au lt R . L ., Ed ge S . B ., Won g Y . N. , et al . (2012). C li nicopathol o gic featur es, patt erns of recurr enc e, and survival among women with tripl e-negati ve bre ast cance r in the Nati onal Comprehensive Cancer Netwo rk. Cancer , doi: 10.1002/cnc r.27581. Liu J ., Deng H ., J ia W ., Zen g Y ., Ra o N ., Li S . , et al . (2012). Comparison of ER / P R and HER2 statuses in primar y and paired liver met astatic sit es of bre ast carcin oma in pati ents with or without treatm ent. J Cancer Res Clin Oncol, Low er E.E., Glass E. L., Bradl e y D.A., Blau R. and Hef felfin ge r S. (20 05). Impact of metastatic estro gen rec ep tor and pro gesteron e rec eptor status on survival. Breast Cancer Res. Treat , 90 : 65-70. Maki D.D and Grossm an R. I. (2000). Patt erns of disease in metastatic bre ast carcinom a: In fluenc e of estro gen an d progesterone rec eptor status. Am J Neuro Radiol , 21: 1064 - 1066. Margolese R. G, Fisher B. , Hortob a g yi G. N. and Bloom er W . D: Neoplas ms of the breast. Ontario: Decke r B.C. (2000 ). In Cancer Medicine . (18 )15. University of Ghana http://ugspace.ug.edu.gh 65 Marshall L.M., Hunter D.J ., Connoll y J . L., Sch ni t t S.J ., B yrne C., Lon don S.J ., et al. (1997). Risk of bre ast cancer associated with at yp ic al hyp erplasia of lob ular and duct al t ypes. Cancer Epidemiol Biomarkers Prev., 6 (5): 297 -301. Maurer H.R. and Chalkl e y G.R. (1974). Some properties of a nuclea r bindi ng sit e of estradiol . J Mol Biol , 27: 431 -441. McCart y K.S. (1977). Steroid hormone rec eptors in the regulation of diffe renti ati on. Am J Pathol , 86 : 705. Mitra A.B. , Murt y V.V .S . and Pratap M. (199 4) . ERBB2 (HER2/ neu) oncogene is frequentl y ampl ified in squamous cell car cinom a of the uterine ce rvix . Cancer Res , 54 : 637- 639 . Moasser M. M., Basso A., Averbuch S.D. and Rosen N. (2001). The Tyr osine Kinase Inhibitor =D1839 (³Iressa´) Inhibits HER2-drive n Signali ng and Suppres ses the Growth of HER2-over ex pressi ng Tumor Cell s. Cancer Res , 61 : 7184. Molina M.A , Sáez R ., Ramse y E.E ., Gar cia - Ba rchino M.J , Rojo F ., Ev ans A.J ., et al . (2002). NH (2 )-te rminal truncate d HER-2 pr otein but not full -length receptor is associated with nodal metastasis in human breast canc er. Clin Cancer Res., 8 (2): 347-3 53. University of Ghana http://ugspace.ug.edu.gh 66 Moriart y K., Kim K.H. and Bender J .R. (2006 ). Mi nireview : Estro gen re ceptor - medi ated rapid sign ali ng . Endocrinology, 147 : 5557 ±556 3 . Mori I., Yan g Q. and Kakudo K. (2002). Pr edictive and pro gnost ic m arke rs for invasive breast cance r. Pathol Int , 52: 186-194. Mote P.A ., Bartow S ., Tr an N . and Clarke C. L . (2 002). Loss of co-ordinat e ex pressi on of progesterone recepto rs A and B is an earl y ev ent in breast carcino gen esis . Breast Cancer Research and Treatment, 72(2): 163-172. Muss H.B., Case D. and Richards F. (1991). Inte r rupted versus conti nuous chemot hera p y in patients with metastatic breast canc er. N Engl J Med, 325: 1342 -1348. Nagao T ., Kinoshi ta T ., Hojo T ., Tsuda H ., Tamura K . and Fuji war a Y . (2012). The differen ces in the hist ologic al t ypes of br east cancer and the response to neoadjuvant chemot herap y: The rel ati onshi p between the outcome and th e cli nicopathol ogic al chara cterist ics. Breast , 21 (3): 289 -2 95. Nahta R ., Yuan L. X ., Zhan g B ., Koba ya shi R . and Esteva F.J . (2005). Insul in -li ke gro wth factor - I rec eptor /hum an epidermal growt h factor rec eptor 2 hete rodimeriz ati on contribut es to trastuz umab resis tance of bre ast ca ncer cell s. Cancer Res . , 65 (23): 11118 - 11128. University of Ghana http://ugspace.ug.edu.gh 67 Nahta R., Yu D., Hun g M.C ., Hortobag yi G. N. and Esteva F.J . (2006). Mechanism s of disease: understandin g resis tance to HER2 -ta r get ed therap y in huma n bre ast canc er. Nat Clin Pract Oncol ., 3( 5): 269 ±280. Nati onal Tox icology P rogram. Depa rtment of Healt h and Human Services. Press Releases 2002. htt p:/ /nt p.niehs.nih.gov/ go/35330. Access ed 28/3/ 2012. Neder gaard L., Haerslev T. and Jacobsen G.K. (1995). Imm unohis toche mi cal stud y of estrogen receptors in prim ar y bre ast ca rcinom as and their l ymph no de metastases including compa rison of two monoclonal antibodi es. APMIS, 103(1 ): 20 - 2 4. Nilss on S., Makela S. , Treuter E., Tuja gue M., Thoms en J ., Andersson G., et al . ( 2001 ) . Mechanism s of estro gen acti on. Physiol Rev, 81 : 1 535 ±1565. No gu chi M., Ko yas aki M. and Ohta N. (1992). c-erb B-2 oncop rotein ex pressi on versus int ernal mammar y l ymp h node metastases as addi ti onal prognost ic fact ors in pati ents with ax il lar y l ymph node -posi ti ve breast cance r. Cancer , 69 : 2953 -2960. Okta y M.H ., Gertle r F. B ., Liu Y.F ., Rohan T.E ., Condeeli s J .S . and J ones J G . (2012). Correlated immunohi stochemi cal and c ytol o gic al assa ys for the prediction of hematogenous dissemina ti on of breast can ce r . J Histochem Cytochem., 60 ( 3): 168 -173. University of Ghana http://ugspace.ug.edu.gh 68 Ola yio ye M.A. (2001). Update on HER-2 as a target for canc er ther ap y: int rac ell ular signali n g pathw a ys of ErbB2/HER -2 and fami l y members. Breast Cancer Research , 3 (6): 385 ±389. Osborne C.K., Schiff R ., Arpino G ., Lee A.S . and Hilsenbeck V.G . (2005). Endocrine responsi veness: unde rsta nding how pro gest erone r ecepto r can be used to select endo crine therap y. Breast , 14 (6 ): 458-465. Parise C.A ., Bau er K.R ., Brown M.M . and Ca ggi ano V . (2009). Breast ca ncer subt ypes as defined b y the estro gen rec eptor ( ER ), pro ge sterone receptor ( PR ), and the human epidermal growth factor recepto r 2 (HER2 ) amon g women with invasive breast can ce r in Cali fornia, 1999-2004. Breast J, 15(6): 593 -602. Paluch-S him on S., Ben - Baru ch N., Wolf I., Zac h L., Kru gli kova A., La ndau E., et al . (2005). Breast canc er with HER2/neu over -ex pressi on and hormone receptor posi ti ve status: - A dist inct biol ogy and natural histor y. J Clin Oncol., 23(16): 650. Perez E.A., Romond E. H. and Suman V.J . (20 07). Updated result s of the combi ned anal ysis of NCC TG N9831 and NSABP B -3 1 adjuvant chemot her ap y with/ without trastuz umab in pati ents with HER2 -posi ti ve breast can ce r. J Clin Oncol ., 25 (18S): Abstract 512. University of Ghana http://ugspace.ug.edu.gh 69 Perou C.M ., Sørli e T ., Ei sen M.B ., van de Rij n M ., J effre y S.S ., Rees C.A . et al . (2000 ). Molecular portr ait s of hu man breast tumours. Nature, 406 (6797): 747-752. Popescu N.C., King C.R. and Kraus M.H. (1989). Locali z ati on of the human erbB-2 gen e on normal and rear ran ged ch romosomes 17 to bands q12 -21.32. Genomics, 4 : 362 - 366. Prat A. and Perou C.M. (2009). Mammar y dev elopement meet can c er genomi cs. Nat Med., 5: 842 -844. Rack B., Schindl be ck C., Ander gass en U., Lor enz R., Zwin ge rs T., Schnee weiss A. , et al . (2010). Prognost ic role of circulatin g tum or cell s in the peripheral blood of breas t canc er pati ents. Cancer Res ., 70 , 93. Rais G., Raiss ouni S ., Ait elhaj M ., Rais F ., Naci ri S ., Kho ya ali S ., et al .(2012). Tripl e negat ive br east canc er in Morocc an women: cli nicopathol ogi cal and thera peuti c stud y at the Nati onal Insti tut e of Oncolog y. BMC Womens Health, 12:35 . Rambau P. F., Chal ya P . L., Man yama M. M. and Jackson K. J. (2011). ³Pathological featur es of Breast Canc er see n in Northweste rn Tanz ania: A nine ye ar s retrospecti ve study´ BMC Research Notes , p. 214 . University of Ghana http://ugspace.ug.edu.gh 70 Rampaul R.S ., Pinder S. E., Elaston C.W . and Elli s I.O. (2001). Pro gnost ic and predictive facto rs in prim ar y br east cancer and their role in pati ent management; the Notti ngham breast team. Eur J Surg Oncol , 27: 229 -2 38 . Rastelli F . and Crispino S . (2008). Facto rs predic ti ve of response to horm one therap y in breast cance r. Tumori , 94 (3): 370 -3 83. Reichelt U. , Duesed au P. and Tsourlakis M. C h . ( 2007). Frequent homo genous HER -2 ampli ficati on in prim ar y and met astatic ad enoc a rcinom as of the esopha gus . Mod Path , 20(1 ): 120-129. R iggins R. B ., Bouton A.H ., Liu M.C . and Clark e R . (2005). Antiestro ge ns, a romatase inhi bit ors, and apoptos is in breast can cer. Vitam Horm , 71: 201 -237. Rubin I. and Yard en Y. (2001). The basic biol ogy of HER2. Ann Oncol , 12(suppl1):S 3 - S 8. Ruh T.S., Katz enell enbo gen B.S . and Gorski J . (1973). Estrone int era cti on with the rat u terus: In vit ro response and nuclea r uptake. Endocrinology, 92 : 125. Saigal K., et al . (2011). Pathol ogic resp onse and locore gional outcome in post menopausal ER posi ti ve breast canc er pati ents receivi n g neoadjuv ant endocrine therap y vs c ytot ox ic che mot her ap y. ASTRO, abstr act 181. University of Ghana http://ugspace.ug.edu.gh 71 Sari E ., Guler G ., Ha yra n M ., Gullu I ., Altunda g K . and Ozisi k Y . (2011 ). C omparati ve stud y of the immunohi s tochemi cal dete cti on of hormone receptor statu s and HER - 2 ex pressi on in prim ar y and paired recur rent/ met astatic lesions of pa ti en ts with breast canc er. Med Oncol , 28 (1 ): 57 -63. Scalt riti M ., Rojo F ., Ocaña A ., Anido J ., Guz man M ., Cortes J ., et al . (2007). Ex pressi on of p95HER 2, a truncated form of the HER2 recepto r, and response to anti -HER2 th erapies in bre ast can cer . J Natl Cancer Inst . , 99 (8):628 -6 38. Schlessinge r J. (2000). Cell signali n g b y receptor t yr osine kin ases. Cell , 103: 211 -225. Schuell B. , Gruenbe r ger T. and Scheit hau er W. (2006). HER 2/neu prot ein ex pressi on in colorectal canc er . BMC Cancer , 8 (6 ): 123 . Science in Afric a (2005). Breast can ce r findings in Afric an women. htt p:/ /www.scienceinaf rica.co.z a/2005/ april /bre as tcance r.htm . Date last ac cessed 13/5/12 Seshadri R., Firgaira F. A. and Horsfall D.J . (1993). Cli nical significanc e of HER -2/neu oncogene ampl ific ati on in prim ar y br ea st can cer : The South Australi an Breast Cance r Stud y Group. J Clin Oncol, 11: 1936 ±1942. Shi L., Don g B. and Li Z. (2009). Ex pressi on or ER - Į (36), a Nov e l Variati on of Estrogen R eceptor. JCO, 27 (21): 3413 -3415. University of Ghana http://ugspace.ug.edu.gh 72 Shou J ., Massarweh S ., Osborne C.K ., Wakeli ng A.E ., Ali S ., Weiss H . , et al . (2004). Mechanism s of tamox ife n resis tanc e: incre ased es trogen receptor - HER2 /ne u cross - talk in ER / HER2 - posi ti ve breast canc er. J Natl Cancer Inst. , 96 (12): 926-935. Simmons C ., Mil ler N ., Geddie W ., Gianfeli ce D ., Oldfield M ., Dranit saris G . , et al. (2009). Does confirm ator y tum or biops y alt er th e mana gem ent of bre ast cancer pati ents with dist ant metastases? Ann Oncol ., 20(9): 1499 - 1 504. Sim pson J .F., Gra y R., Dressl er L.G., Cobau C.D., Falkson C. I., Gilchri st K.W . , et al. (2000). Prognost ic value of hist ologic gr ade and proli ferati ve acti vit y in ax il lar y nod e - posi ti ve breast canc er: re sult s from the East ern C ooperati ve Oncolo g y Gr oup Companion Stud y, EST 4189. J Clin Oncol , 18 : 2059 -20 69. Sim pson P.T., Reis -Fil ho J .S , Gale T. and Lakh ani S.R. (2005). Molecular Evolut ion of Breast Cance r. J Pathol, 205: 248 ±254. Slamon D.J . Clark G.M. and Wong S.G. (1987 ). Human bre ast can cer: correl ati on of relapse and survival wit h ampl ificati on of HER -2/ neu onco gen e. Science, 235 : 177 -182. Smith I., Procte r M. and Gelber R.D. (2007). 2- ye ar follow -up of tras tuz umab after adjuvant ch emot herap y i n HER2 -posi ti ve br east canc er: a randomi sed co ntroll ed trial. Lancet ., 369 (9555): 29 ±3 6. Sobin L.H. (2003). TN M: evolut ion and relation to other prognost ic fac tors. Semin Surg Oncol. , 21(1): 3 -7. University of Ghana http://ugspace.ug.edu.gh 73 Song R.X and Santen R.J . (2006). Membrane ini ti ated estrogen sign ali ng i n breast canc er. Biol Reprod ., 75(1): 9 -16 . Stahlberg C., Ped ersen A.T., Lyn ge E., Anders en Z.J ., Keidi ng N., Hund ru p Y.A., et al . (2004). In cre ased ris k of breast can cer following diffe rent regim ens of horm one replac ement therap y freq uentl y used in Europ e . Int J Cancer , 109 : 721 -727 . Stark A., Kleer C.G ., Marti n I ., Awu ah B ., Nsia h -Asar e A ., Tak yi V . , et al . (2010). African anc estr y and hi gher prev alenc e of tripl e- negati ve bre ast can cer : fi ndings from an int ernati onal st ud y. Cancer . , 116 (21):4926 -3 2. Stead L.A., Lash T. L., S obieraj J .E., C hi D.D., Westrup J .L., Charlot M., et al . (2009). Tripl e-ne gati ve breast ca ncers are incr eas ed in black women re gardl ess of age or bod y mass index . Breast Cancer Research, 11: R 18 . Stevens K.N., Fr ede ri cks en Z., Vachon C.M., Wa ng X. and Mar goli n S. (2012). 19p13.1 Is a Triple -N e gati ve-S p ecific Br east Can cer Suscepti bil it y Locus. Cancer Res ., 72 (7): 1795-1803. Struewing J .P ., Hartge P . and Wachold er S. (199 7). The risk of can ce r as sociated with specific mut ati ons of BR CA1 and BRCA2 amon g Ashken az i J ews. N Engl J Med , 336 : 1401- 1408. University of Ghana http://ugspace.ug.edu.gh 74 Su D ., Fu X ., Fan S ., Wu X ., Wang X.X ., Fu L ., et al . (2012). R ole of ERR F, a nov el ER - related nucle ar facto r, in the growth control of ER -posi ti ve human breast cancer cell s. Am J Pathol, 180 (3): 1189 -1 201. Tanner M., J arvinen P. and Isol a J . (2001). Ampli ficat ion of HER-2/neu and topoi somerase II alpha in prim ar y and metastati c breast canc er. Cancer Res , 61 : 5345- 5348. Troester M.A. and Swift -S canlan T. (2009 ). Chall enges in stud yin g th e eti olog y of br east canc er subt ypes. Breast Cancer Research , (edit or ial ) 11: 104 Umekit a Y., Sa gar a Y . and Yoshida H . (1998 ). Estrogen receptor mut ati ons and chan ge s in estrogen receptor an d progesteron e receptor protein ex pressi on in metastatic or recur rent br east can ce r. Jpn J Cancer Res , 89 (1): 27 -32. Vogel C. L., Cobleigh M .A. and Tripath y D. (20 02). Effica c y and safet y of trastuz umab as a single agent in first - li ne treatm ent of HER2 -overex press ing metast ati c breast can cer. J Clin Oncol ., 20(3): 719 ±726. Wei L. L., Gonz alez -Alle r C., Wood W.M., Miller L.A. and Ho rwit z K.B. (1990). 5' - Hetero gen eit y in human progester one rec eptor transcript s predicts a new ami no - termi nal truncated "C "- r eceptor and unique A-r e ceptor messa ges. Mol Endocrinol , 4: 1833 -1840. University of Ghana http://ugspace.ug.edu.gh 75 Whelan T.J ., J uli an J ., Wright J ., J adad A.R. an d Levine M. L. (2000 ). Does locore gional radiation therap y impro ve survival in breast cancer? A meta -anal ysis . J Clin Oncol , 18(6): 1220 -1229. W il li ams D. and Gorski J . (1974). Equil ibrium bindi ng of estr adiol by ute rine cell suspensions and whole uteri in vitro. Biochemistry , 13 : 5537 -5542. Wiredu E.K . and Arm a h H.B. (2006). C ance r mortali t y patt e rns in Gh ana: a 10 - ye ar review of autops ies and hospi tal mortali t y. BMC Public Health , 6 : 159. Yardle y D. A. (2000 ). In Pursuit of the Preventi on of Breast Canc er. Am J Med Sci, 320(4): 263-272. Yan g Q., Khour y M.J . and Rodriguez C. (1998). Fami l y hist or y scor e as a predictor of breast can cer mortali t y: prospecti ve data from the Cancer Prev enti on Stud y II, United States, 1982 -1991. Am J Epidemiol , 147 (7): 652 - 659. Zidan J ., Dashkovsk y I., Sta yerman C., Basher W., Coz acov C. and Hadar y A. (2005). Comparison of HER -2 overex pressi on in prim ar y breast can ce r and metas tatic sit es and its effect on biol ogic al targeti ng the rap y of metast ati c disease. Br J Cancer. , 93 (5): 552 ± 556. University of Ghana http://ugspace.ug.edu.gh 76 Zhan g Y., Hen g-Yin Y., Xiao -C hun Z., Yan g H., Tsa i M. and Mon g -Hon g. (2004). Tumor suppressor AR F inhi bit s HER -2/neu-mediated onco geni c growth. Oncogene, 23: 7132± 7143. University of Ghana http://ugspace.ug.edu.gh 77 APPENDIX I DESC R IP T ION OF ANT IBO D IES. ESTR OGEN RECEP TOR: Product Code: NC L- L- E R -6F11 LOT: L1151211 Clone: 6F11 Imm un o gen : Proka r yoti c recombi nant prot ein correspondi n g to the full length alpha form of the human estro gen rec eptor molecule. Imm uno globui n: Mouse monoclonal Imm uno globul in class: IgG1 Antibod y con centrati on: Greate r than or equal to 67.5mg/ L as det ermined by ELIS A Total protein conc entrati on: 4.1g/ L Total antibod y con centr a ti on: 75m g/ L Dilut ion factor of anti bo d y used: 1/60 Concentrati on of anti bod y used: 75m g/ L X 1/60 = 1.25mg/ L PROGESTER ONE RECEPTOR : Product Code: NC L- L-P GR -312 LOT: 6008675 Clone: 16 Imm uno gen : Proka r yoti c recombi nant prot ein correspondi n g to the N -te r mi nal region of the human pro geste ro ne rec eptor molecule. Imm uno globui n: Mouse monoclonal Imm uno globul in class: IgG1 Antibod y con centrati on: Greate r than or equal to 342.0mg/ L as det er mi ne d by ELIS A Total protein conc entrati on: 4.6g/ L University of Ghana http://ugspace.ug.edu.gh 78 Total antibod y con centr a ti on: 360m g/ L Dilut ion factor of anti bo d y used: 1/100 Concentrati on of anti bod y used: 360m g/ L X 1/10 0 = 3.6mg/ L HUMAN EP IDERMA L GROW TH FACTOR RECEP TOR -2: Product Code: NC L- L-C B11 LOT: 6009335 Clone: CB11 Imm uno gen : S ynthetic pepti de corr espondi ng to a sit e on the internal dom ain of the human c-e rbB -2 oncopro tein. Imm uno globul in class: IgG1 Antibod y con centrati on: Greate r than or equal to 23.4mg/ L as det ermined by ELIS A Total protein conc entrati on: 3.9g/ L Total antibod y con centr a ti on: 26m g/ L Dilut ion factor of anti bo d y used: 1/40 Concentrati on of anti bod y used: 26m g/ L X 1/40 = 0.65mg/ L University of Ghana http://ugspace.ug.edu.gh 79 APPENDIX II R EAGENT PREPAR ATIO N 50mM Tris Buff ered Sali ne (TBS ): The foll owing wer e diss olved in 800ml of disti ll ed water; 8g Sodium chloride 0.2g potassium chloride 6g Tris Bas e PH was then adjust ed to 7.6 with concentrat ed h ydrochloric acid Total vol ume was made up to 1 L with disti ll ed water. 0.01M Citric acid: 2.1g of cit ric acid was di ssol ved in 1 L of disti ll ed water PH was adjusted to 6.0 University of Ghana http://ugspace.ug.edu.gh 80 APPENDIX III University of Ghana http://ugspace.ug.edu.gh 81 University of Ghana http://ugspace.ug.edu.gh