Investigation of gene amplification and expression in prostate cancer

LEABHARLANN CHOLAISTE NA TRIONOIDE, BAILE ATHA CLIATH TRINITY COLLEGE LIBRARY DUBLIN OUscoil Atha Cliath The University o f Dublin. Terms and Conditions of Use of Digitised Theses from Trinity College Library Dublin. Copyright statement. All material supplied by Trinity College Library is protected by copyright (under the Copyright and Related Rights Act, 2000 as amended) and other relevant Intellectual Property Rights. By accessing and using a Digitised Thesis from Trinity College Library you acknowledge that all Intellectual Property Rights in any Works supplied are the sole and exclusive property of the copyright and/or other I PR holder. Specific copyright holders may not be explicitly identified. Use of materials from other sources within a thesis should not be construed as a claim over them.. 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Electronic or print copies may not be offered, whether for sale or otherwise to anyone. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.. INVESTIGATION OF GENE. AMPLIFICATION AND EXPRESSION IN. PROSTATE CANCER. f ^ T R I N I T Y C O L L E G ^. n 5 O C T 2fiQ7. LIBRARY DUBLIN ^. Declaration. a) T his thesis has not been su bm itted p reviously fo r a degree at this or any other. university.. b) T h is thesis is m y ow n w ork how ev er the con trib u tio n o f Dr. A m anda M urphy w ho. co n stru c te d the tissue m icroarrays m ust be acknow ledged.. c) 1 agree that the library m ay lend or copy the thesis upon request.. i | - ^ . , . ' • ■ ^ > ‘‘ ^»..'î ''r‘3 -- --‘ ^ - j ^ ' ' • •• r ^ > ■ » ' • > * ’ - • • - • •. L'"’-1̂-. ; N&<r̂ ■'. ■ ••;ngL<«rf^ \ ‘ ^ ’ ^ • - 5 ^ . r'C ^.r-'- ■ • • jV * .'. *-'WV-. ■" • , T ^ iL v t- ,'iT i:- ''/^ i.i.i. ‘amS^i.^W'fcLw. -.J?. ■. .y'i- ̂ j ....... V r “ , ". l-fVy-T’ . ''*• , ,1 ■>.! > " • < .. t f i : ? " " : • :. f t * . "4jSSk. ' , Uj ;/-i-st.i.. ‘ - . i ; J i i * t , ^ •• i> ' . -w -', '.-T - : -•. ■ • , ; V ■ ■. ; J. 3f • M -V" " • - V - # '•” !' 'W ' » i i« • f l. l i ' SiijfeeiiiMVV. ■■î. Summary. The purpose o f this thesis w as to identify significant genetic am p lifica tio n s and protein. expression o f a gene o r genes th at have not been recognised as o v e ra m p lifie d to date in. prostate cancer.. M ethods used:. 1) Investigation o f po ssib le gen e am plifications in p rostate cancer:. Laser capture m icro d issectio n w as used to identify/capture m alig n a n t glands from three. tissue sam ples o f prostate c a n c e r and benign glands from o n e c ase o f benign prostatic. hyperplasia. By u tilisin g this tec h n o lo g y w e could ensure that w e w ere ex am in in g genetic. changes in epithelial cells o n ly and thus avoid the b ack g ro u n d n o ise o f genetic changes. v/ithin the strom a.. DNA w as extracted from the m icro d issected tissue and a m p lifica tio n o f the extracted. DNA was perform ed by D O P -P C R .. CG H m icroarray analysis w as u se d to detect possible gene am p lifica tio n s w ithin the. DNA o f the sam ples.. M ajor findings in the in v estig a tio n o f possible gene a m p lifica tio n s in prostate cancer. using C G H m icroarray analysis:. The gene T opoisom erase II a lp h a gen e w hich is in v o lv ed in cell rep lication was. s.gnificantly am plified in all o u r p rostate can cer sam ples. T h is gen e w as am plified to the. greatest extent in the case o f a d v a n ce d horm one in se n sitiv e p ro sta te cancer.. 2» Identification o f pro te in o v e re x p re ssio n o f T o p o iso m e rase II a lp h a gene in prostate. cincer:. T he second m ajor area o f in v estig a tio n o f this thesis w as to identify w hether there was. sig n ifican t protein ov erex p ressio n o f T o poisom erase II alpha gene in a larger cohort o f. prostate cancer patients. T h is w as a c h iev ed by an im m unohistochem ical study using 100. prostate can cer sam ples and 42 c o n tro ls (benign prosatic hyperplasia).. T he results o f this study w ere a n a ly se d by the SPSS statistical pack ag e using the U M ann. W hitn ey test.. M ajor findings o f the im m u n o h isto ch em ical study:. T here w as no statistically sig n ific a n t difference betw een the g roups benign prostatic. hy p erp lasia and prostate c a rc in o m a G leason Score 6 (P= 0.245). T h e d ifference betw een. the g ro u p ’s prostate carcin o m a G le a so n Scores 6 and 7 was statistically significant. (P < 0.008). In addition there w as a h ig h ly statistically significant d ifferen ce betw een the. groups prostate carcinom a G le a so n S core 7 and G leason scores 8,9 and 10 (not selected. for horm onal status), (P = 0.000).. W hen the category o f G leaso n sco re 8,9,10 was analysed as reg ard to horm one treatm ent. and insensitivity (n=15) and c ases w ith no horm one treatm ent (n= 34) the difference in. T o p o iso m erase indices ap p ro a c h e d statistical significance (P = 0.081).. C onclusion:. T h ere w as statistically sig n ific a n t o v erex p ressio n o f T o p o iso m erase II alpha gene in the. high G leaso n score prostate ca n ce rs co m p ared to low er G leason score prostate cancers. and benign prostatic h yperplasia. T h e se findings indicate that T o p o ism e rase II alpha gene. is a potential gene target for c h e m o th e ra p y in high G leason score p rostate cancers.. CHAPTER ONE. GENERAL INTRODUCTION. 1. 1.1 In trod u ction. T h is study investigates gene am p lifica tio n and expression in prostate cancer.. In this introductory ch ap ter w e sh all review w hat is know n reg a rd in g genetic changes in. p ro state cancer.. P rostate cancer is the second lea d in g cause o f can cer deaths in m en. It is not invariably. leth al, how ever and is a h e te ro g en e o u s disease ranging from asym p to m atic to a rapidly. fatal system ic m alignancy. T h e p rev a len c e o f p rostate c a n ce r is so high that it could be. c o n sid e red a norm al age related phenom enon. 33% o f m ales in th eir 8‘̂ decade had. e v id e n c e o f prostate cancer at a u to p sy and died w ith the disease but not from it in a. S panish study. This study e x a m in e d C aucasian M editerranean m ales and data from. A m erican autopsy studies show an even h ig h er prostate c a n c e r prev alen ce rate. [ 1 ] In. recen t years there have been b ig increases in the five year survival rates for prostate. c a n c e r w ith a 5 year relative age stan d ard ised survival rate o f 65% in E ngland and. W ales for the years 1996-1999. T h is w as the third highest survival rate o f all cancers. o v e r this tim e period w ith o n ly testic u lar c a n ce r and m elan o m a hav in g superior survival. rate s and was around 11% h ig h e r than that fo r p atients d iagnosed du rin g 1991-1995.[2]. T h is im provem ent does not u n fo rtu n a te ly reflect b e tte r treatm en t for prostate cancer. It. larg e ly reflects an increasin g n u m b e r o f m en bein g d iag n o sed w ith very early stage. p ro sta te can cer as a resu lt o f w id e sp re a d use o f p rostate sp ecific antigen (PSA ) testing.. P S A is a protein produced b y b o th norm al and cancerous p ro sta te cells and a high PSA . level can be a sign o f cancer. M o st m en d iagnosed at a very early stage will die w ith. 2. p ro sta te cancer but not from it, th ere fo re the survival rate has increased. T he A m erican. C a n c e r Society recom m ends in its ’ prostate cancer screen in g guid elin es that m en be. inform ed on w hat is know n and w h at is uncertain about the b enefits and lim itations o f. e a rly detection o f prostate c a n c e r so that they can m ake an in fo rm e d decision about. testing. T herefore the early d iag n o sis o f prostate can cer th ro u g h screening creates. d ifficu lties in predicting the o u tc o m e o f individual patients. T h e difficulty is in. d istin g u ish in g betw een c lin ic a lly indo len t prostate cancers, w hich w ill be. asym p to m atic, and agg ressiv e p ro sta te cancers w ith the po ten tial to kill the patient.. G le a so n grading on h isto p a th o lo g ic a l exam ination is the best prognostic indicator to. d ate in prostate can cer h o w e v e r in te r-o b se rv e r variation can o c c u r, grading on biopsies. m ay not correlate w ith the p ro sta te c to m y specim en due to sam p lin g problem s and cases. o f m o rphologically identical p ro sta te cancer can behave differen tly .. 1.2 New Technologies. T h is is an exciting era w ith the e m e rg en c e o f new in v estig a tiv e tools such as D N A. m icro array technology and the applicatio n o f the field o f p ro te o m ic s to the study o f. h u m an cancers. K now ledge o f g e n etic changes u n d erly in g initiatio n , developm ent and. p ro g ressio n o f p rostate ca n ce r is a c cu m u latin g rapidly. W ith in creasin g know ledge it. m ay be possible to d istinguish in d o le n t from a g g ressiv e p ro state tum ours by m olecular. fingerprinting. A clinical a p p lic atio n o f this know ledge w o u ld be that radical treatm ent. and its ’ associated m orbidity c o u ld be avoided in prostate cancers unlik ely to progress.. R eso u rces and radical treatm en t co u ld be focussed on p ro sta te c an cers w ith p oor. p ro g n o stic indicators. W ithin this general introduction c h a p te r w e shall discuss the. 3. m ost co n sisten tly reported m olecular p athological findings in prostate can cer together. w ith new co ncepts and technologies.. 1.3 Hereditary Prostate Cancer. Prostate c a n ce r can be divided e p id e m io lo g ic a lly into hereditary and sporadic form s[3]. but it is not possible to distinguish th ese 2 groups on a m olecular level. H ighly. p en etran t inherited genes co n ferrin g the p ro state cancer phenotype hav e not been. identified.. L inkage studies using genetic m ark ers to search for chrom osom al reg io n s that show. e x c essiv e sharing o f inherited alleles in c a n ce r fam ilies have been helpful in identifying. im portant c a n ce r susceptibility genes in o th e r cancers. H ow ever sim ila r studies using. p ro state c a n ce r fam ilies have not yield ed the sam e success.. A lthough possible inherited prostate c a n c e r susceptibility genes have been identified. such as E L A C 2, R N A SE L, M S R l, N S B l and C H E K 2 genes in som e fam ilies, the. pro p o rtio n o f cases o f hereditary p ro state c a n c e r attributable to g erm lin e m utations in. these loci is sm all. M any studies hav e not supported the role o f th ese genes in. h ered itary prostate cancer. M utations o f th ese candidate genes have also been identified. in sporadic p rostate cancer. As p ro state c a n c e r is a com m on can cer it m ay be difficult. to d istin g u ish clustering o f sporadic p ro sta te can cer w ithin fam ilies fro m true hereditary. p ro state cancer. T his difficulty m ay hav e hin d ered research in h ered itary prostate. c a n c e r to date. A lternatively the failu re to identify highly penetrant genes in hereditary. p ro state c a n c e r m ay be due to the fac t th at m u ltiple genes w ith sm all to m oderate effect. are in v o lv ed in hereditary prostate carc in o g e n e sis. The risk o f d isease in the presence o f. 4. a su sce p tib ility gene m ight be su b sta n tia lly increased o n ly in the a p p ro p riate genetic,. d ietary and en v ironm ental b a c k g ro u n d .[4] W e will b riefly outlin e the m ost significant. h ered itary p ro state can cer su sceptibility g en es to date.. 1.3.1 P ossible Cancer Susceptibility G enes. E L A C 2. E L A C 2 w as the first identified po ssib le h ered itary p rostate ca n ce r gene. T h e function. o f E L A C 2 is not definitively know n and it has been pro p o sed as a m etal dependent. hydrolase. A ssociation o f E L A C 2 g en o ty p e s w ith fam ilial p ro state c a n c e r have been. reported. [5]. H o w e v er m ultiple large subsequent stu d ie s have not p rovided c o n firm a to ry evidence o f. this a sso c ia tio n .[6 ][7 |. O verall it appears that if E LA C 2 plays a role in prostate can cer it is a w e a k role.. H ost R esp o n se to Infection G enes;. R N A S E L. R N A S E L is a ribonuclease that d eg rad es viral and cellu lar R N A and can produce. ap o p to sis on viral infection. M u tations in the R N A S E L gene have been identified in. fam ilial and sporadic prostate c a n c e r in m u ltip le stud ies.[8 ][9 ][1 0 ][1 1 ][1 2 ] O ther. studies have not supported these fin d in g s .[1 3 ][I4 ]. O v erall there is strong support that R N A S E L is the m ost im portant h e re d ita ry prostate. c a n c e r g e n e id en tified to date.. 5. M S R l. M S R l, enco d es a m acrophage scavenger re c e p to r resp o n sib le for cellular u p tak e o f . m olecules in clu d in g bacterial cell wall products. T h e im p o rtan ce o f M S R l as a prostate. c a n ce r su sce p tib ility gene in hereditary p ro state c a n c e r is controversial. G erm lin e. M S R l m utatio n s hav e been linked to prostate c a n c e r in som e fam ilies w ith p ro state. can cer and in sporadic prostate c a n ce r.[15][16] H o w e v er a recen t report, w h ich . investigated 163 fam ilies w ith fam ilial p rostate can cer, did not provide c o n firm ato ry . evidence o f the ro le o f M S R l in fam ilial p ro sta te cancer.[17]. Possible m ec h a n ism s o f action by w hich m u tatio n s o f these host response to infection. genes increase the risk o f prostate cancer is that th ey m ay predispose to ch ro n ic. inflam m ation due to failure o f viral RN A and ba c te ria l d egradation. T here is. accum ulating k now ledge supporting the role o f in fla m m atio n in prostate can cer, w hich. we will refer to again later in the article.. C ell C ycle C h e c k p o in t G enes:. N B S l. T he rare hum an g e n etic disorder, N ijm egen B re a k a g e S y n d ro m e, is c h a ra c te rise d by . radio sen sitiv ity , im m unodeficiency, c h ro m o so m a l in sta b ility and an in creased risk for. 6. c a n ce r o f the ly m p h atic system . T he N B S l gen e w h ich is involved in this hu m an . genetic disorder, codes for a protein, nibrin, in v o lv ed in the p ro ce ssin g /rep a ir o f D N A . double strand break s and in cell cycle c h eck p o in ts. [18] M utations in the gene fo r the. N ijm egen b rea k a g e syndrom e (N B S l) have been id en tified in both sporadic and. fam ilial cases o f p ro state cancer and are a sso c ia te d w ith a sm all in creased risk o f . p rostate cancer. [19]. C H E K 2. T he C H E K 2 gene is an upstream regulator o f p53 in the D N A -d a m a g e -sig n a llin g . pathw ay. C H E K 2 m utations have been id en tified in b o th sporadic and fam ilial cases o f. p ro state ca n ce r and are associated w ith a sm all in cre ase d risk o f prostate. cancer.[20][211. N B S 1 and C H E K 2 genes have only recently been id en tified as possib le p ro sta te cancer. su scep tib ility genes. E L A C 2 was the first h ere d ita ry p ro state can cer su sce p tib ility gene. identified and sub seq u en t studies have not p ro v id e d co n firm ato ry e v id en ce o f its ’ role. in p ro state cancer. T h erefo re it is not possib le to c o m m e n t on the im p o rtan ce o f these. tw o genes in h ered itary prostate can cer until a d d itio n a l c o nfirm atory studies h a v e been. perform ed.. 7. Table 1 Hereditary Prostate Cancer Genes. G ene C hrom osom al. locus. Putative fu nction Status in prostate. cancer. ELA C 2 17p M etal d e p e n d en t hydrolase U nknow n. R N A SE L iq R ibonuclease th at degrades. viral and c e llu la r R N A and. can produce a p o p to sis on. viral infection. Deleted. M S R l 8p E ncodes a m acro p h ag e. scavenger rec e p to r. responsible fo r c e llu la r. uptake o f m olecules. including ba c te ria l cell wall. products. Deleted. NBSI 5p Encodes for a protein,. nibrin, in v o lv ed in the. p ro ce ssin g /rep a ir o f D N A . double strand b rea k s and in. cell cycle ch e ck p o in ts. D eleted. C H E K 2 22q U pstream reg u la to r o f p53. in the D N A -dam age-. signalling p a th w a y. D eleted. T h e study o f hereditary p ro state cancer genes is in its ’ in fan cy and the ch allen g e for the. future will be to detect genes o f sm all to m oderate effects. A dvances in statistical. m ethods to am plify sig n als from susceptibility genes in th e presence o f hetero g en eo u s. factors are required in o rd er to d ecipher the genetics a n d m o le c u la r pathology o f. h ereditary prostate cancer.. 1.4 Sporadic Prostate Cancer. T h e vast m ajority o f p ro state can cer is sporadic. In o u r d isc u ssio n o f the m o lecu lar. pathology o f sporadic p ro sta te cancer we will d iscuss the e v id en ce to date un d er the. fo llow ing categories; po ly m o rp h ism s associated w ith in creased prostate c a n ce r risk,. som atic genetic chan g es and factors involved in the p ro g ressio n o f prostate ca n ce r such. as the androgen receptor, g row th factors and invasion a n d m etastasis genes. W e w ill. d iscuss separately recent findings o f gene ov er and u n d e r-e x p re ssio n by m icro array. technology. T he applicatio n o f the field o f p roteom ics to the study o f prostate can cer. and current theories reg a rd in g the role o f inflam m ation in prostate c a n ce r w ill also be. discussed.. 1.4.1 Polymorphisms Associated with Increased Prostate Cancer Risk:. A p olym orphism is a genetic v arian t that appears in at le a st 1 % o f the p opulation.. T h e se com m on genetic po ly m o rp h ism s are likely to h a v e sm all relative risks, yet large. po pulation attributable risks due to their high freq u en cies.. 9. T L R 4. T L R 4 encodes a receptor, w hich is a central player in the sig n a llin g pathw ays o f the. innate im m une response to infection by G ram -negative bacteria. A T L R 4 sequence. p o ly m o rp h ism is associated w ith a sm all increased risk o f p ro state cancer. [22] T his is. in k e e p in g w ith the cu rren t h y p o th esis o f inflam m ation hav in g a ro le in prostate. c arcin o g en esis. W e will discuss the cu rrent hypothesis o f the role o f inflam m ation in. p ro sta te can cer later in the article.. C D K N IB (p27). T h e loss o f cell cycle control is believ ed to be an im portant m ech an ism in the. p ro m o tio n o f carcinogenesis. C D K N IB (p27) belongs to the C ip /K ip fam ily and. fu n ctio n s as an im portant cell cy cle gatekeeper.. A rec e n t study has revealed a sig n ifican t association betw een a single nucleotide. p o ly m o rp h ism o f C D K N IB (p27) and prostate cancer.[23] T h ere is also an association. b etw een C D K N IB (p27) and a n o th e r C ip/K ip fam ily m em ber, C D K N IA (p21) and. a d v a n ce d prostate cancer. [24]. 10. Growth o f prostate cells depends on androgens. Genes that encode products, which play. a role inducing androgen stimulation o f the prostate gland, are very significant. The. androgen receptor (AR) is currently a therapeutic target for the treatment o f prostate. cancer. Other genes involved in androgen stim ulation o f the prostate such as SRD5A2. and CYP17 also hold potential as future therapeutic targets.. Androgen Receptor (AR). The androgen receptor contains polymorphic polyglutam ine (CAG)n trinucleotide. repeats. It has been reported in the past that shortening o f these repeats are associated. with increased prostate cancer risk.[25]. Short CAG length has also been correlated with high grade, high stage, m etastatic and. fatal prostate cancers. A hypothesis that has been proposed for the influence o f the short. CAG repeat on prostate carcinogenesis is that due to its’ role in androgen receptor. function it causes an increase in activation o f androgen dependent genes.[26]. O ther groups have not identified CAG repeats as a risk factor for prostate cancer and a. recent significant study and epidemiological review article have dem onstrated that this. risk factor is less im portant than it has been regarded previously.[27][28]. CYP17. CYP17 encodes cytochrom e P -4 5 0 c l7 a , an enzym e responsible for the biosynthesis o f. testosterone. A variant CYP17 allele is associated w ith both hereditary and sporadic I I. prostate cancer.[29] This allele is hypothesized to increase the rate o f gene. transcription, increase androgen production and thereby increase the risk o f prostate. cancer. [30]. SRD5A2. SRD5A2 encodes the predominant isozym e o f 5-a-reductase in the prostate, an enzyme. that converts testosterone to the more potent dihydrotestosterone. The alleles that. encode enzym es with increased activity have been associated with an increased risk of. prostate cancer and with a poor prognosis for men with prostate cancer.[31][32]. 12. Table 2 Polymorphisms Associated With Increased Prostate Cancer Risk. G ene C hrom osom al. locus. F u n ctio n. T L R 4 9q E ncodes a recep to r w h ic h is a central p lay er in the. signalling pathw ays o f th e in n ate im m une. response to infection by G ra m -n e g a tiv e bacteria. C D K N IB. (p27). 12p B elongs to the C ip /K ip fam ily and functions as an. im portant cell cycle g a te k e e p e r. A R Xq M ay cause activation o f a n d ro g en dependent. genes. C Y P 17 lOq E nzym e responsible fo r th e b io sy n th e sis o f. testosterone.. S R D 5A 2 2p C onverts testosterone to the m o re potent. d ihydrotestosterone. 1.4.2 Polymorphisms associated with Advanced Sporadic Prostate Cancer:. V itam in D R eceptor. Physiologic levels o f vitam in D prom ote the d ifferen tiatio n and grow th a rre st o f. prostate c a n ce r ce lls in vitro.[33] T he precise m ech an ism through w hich vitam in D. m ediates this e ffe c t is unknow n h o w e v e r it is m ost likely through its ’ e ffe c t on cell. grow th proteins. A llelic differences in the vitam in D receptor (V D R ) gene resu lt in. variation in V D R a c tiv ity .[34] V D R alleles have been significantly asso ciated w ith. prostate ca n ce r and this association w as stro n g e r w ith advanced prostate c a n c e rs .[35]. As d iscussed p re v io u sly polym orphism s in C D K N IA (p21‘̂ ''’) and C D K N IB (p27'‘‘'’). are associated w ith ad v an ced prostate c a n c e r.[24] In addition polym orphic v arian ts o f a. num ber o f oth e r g en es have been proposed as p o ssib le contributors to the risk o f. prostate c a n c e r.[36]. Table 3 Polymorphisms Associated With Advanced Prostate Cancer Risk. G ene C hrom osom al locus F unction. V itam in D rec e p to r 13q Prom otes the differen tiatio n . and grow th arrest o f p ro state. c a n ce r cells in vitro. C D K N IA (p21). C D K N IB (p27). 6p. 12p. B elong to the C ip/K ip fam ily . and function as im p o rtan t cell. 14. cycle gatekeepers. 1.5 Somatic Genetic Changes. The num ber o f genetic loci involved in prostate carcinogenesis is large and the. m echanism s are complex and not fully understood. A com pilation o f the most. com m only reported chromosom al abnormalities in sporadic prostate cancer together. with the putative genes involved at these chromosomal sites is presented in Table. 4 .[37][3 8 ]. 15. Table 4: M ost Commonly Described Areas o f Chromosomal Loss and Gain in Prostate Cancer. Chromosome. locus. Putative. genes. Normal function o f gene Status o f gene in. prostate cancer. Ref. No.. 7p EGFR Growth factor Amplified 39. 7q CAV 1 Structural protein o f. caveolae mem branes in. fibroblasts and endothelia. Amplified 40. 8p MSR Encodes a macrophage. scavenger receptor. responsible for cellular. uptake o f molecules. including bacterial cell wall. Deleted 41. 8p NKX3-1 products. Tum our suppressor gene. Deleted 42. 8q c-myc Transcriptional activator Amplified 43. lOq PTEN Tum our suppressor gene Mutated 44. 13q Rb Tum our suppressor gene Deleted 45. 16q E-CAD Adhesion m olecule Deleted 46. Xq AR Androgen receptor Amplified 47. 16. Although these are the most common areas o f chromosom al loss and gain, prostate. carcinogenesis is complex and multiple genes from other chrom osom al loci are also. thought to be involved.. 1.5.1 Tumour Suppressor Genes and Loss o f Heterozygosity. Tum our suppressor genes are likely to be involved in the prostate carcinogenesis. pathway. Loss o f tum our suppressor genes was initially proposed to occur by loss of. function o f two alleles (the “two- hit hypothesis” ) by mutation or d eletio n .[48] This. model has been revised to include epigenetic modification by (a) inactivation o f one or. both alleles by DNA methylation o f CpG sites in gene promoters or (b) function. heritably dow nregulated or (c) otherw ise com prom ised in a clonal fash io n .[49] The. change can be by mutation, methylation o f the prom oter or by some other modification. o f the protein product and must be coupled with evidence that the norm al (w ild type). gene does suppress growth o f tum our cells.[50]. Glutathione S-Transferase Gene (G S T P l). G lutathione S-Transferase Gene (G S T P l) is em erging as one o f the m ost im portant. tum our suppressor genes in prostate cancer. G S T P l can detoxify environm ental. electrophilic carcinogens and oxidants and may have a genome caretaker role by. preventing oxidant and electrophilic DNA dam age. [51] This gene has been shown to be. inactivated by hypermethylation o f the prom oter region in prostate tum ours.[52][53]. H yperm ethylation o f the glutathione S-transferase gene (G S T P l) is the m ost common. 17. (greater than 90%) reported epigenetic alteration in prostate cancer. It occurs early in. cancer progression and it is a promising marker for detecting organ-confined disease.. Quantitation o f G ST P l hyperm ethylation was found to accurately detect the presence. o f cancer even in small, limited tissue samples. It may represent a promising diagnostic. marker that could be used as an adjunct to tissue biopsy as part o f prostate cancer. screening. [54]. Aberrant DNA m ethylation patterns may be the earliest somatic genome changes in. prostate cancer. A recent study found that CpG islands were hypermethylated in >85% . o f prostate cancers and cancer cell lines but not in normal prostate cells and tissues.. CpG island hyperm ethylation patterns in prostate cancer metastases were very sim ilar to. the primary prostate cancers and tended to show greater differences between cases than. between anatomical sites o f m etastasis.[55]. PTEN. PTEN is an important tum our suppressor gene in prostate cancer and influences the. levels o f C D K N IB (p27) another important tum our suppressor gene. The gene for. phosphatase and tensin hom ologue (PTEN), encodes a phosphatase active against both. proteins and lipid substrates and is a common target for somatic alteration during the. progression o f prostate cancer.[56][57] PTEN is present in normal epithelial cells and. in cells in prostatic intraepithelial neoplasia. In prostate cancers the level o f PTEN is. frequently reduced, particularly in cancers o f a high grade or stage. In prostate cancers. that do contain PTEN, a considerable heterogeneity in levels, with regions devoid o f. PTEN has been described.[44] The mechanism by which PTEN might act as a tum our. suppressor gene in the prostate may involve the inhibition o f the phosphatidylinositol. 18. 3’-kinase-protein kinase B (P13K-Akt) signalling pathway that is essential for cell. cycle progression and cell survival.[58]. C D K N IB (p27). C D K N IB (p27) is an im portant tum our suppressor gene in prostate cancer. Reduced. levels o f p27, a cyclin-dependent kinase inhibitor encoded by the C D K N IB gene, are. common in prostate cancers and particularly in prostate cancers with a poor. prognosis.[59] [60] The somatic loss o f DNA sequences at 1 2 p l2 -l, encom passing. C D K N IB , has been described in 23% o f localised prostate cancers, 30% o f m etastases. o f prostate cancer in regional lymph nodes, and 47% o f distant metastases o f prostate. cancer.[61J Levels o f p27 are suppressed by the P I3 K -A k t signalling pathway.[58] By. inhibiting P13K-Akt, PTEN can increase the levels o f C D K N IB m essenger RNA and. p27 protein.[62] For this reason, low p27 levels may be as much a result o f the loss o f. PTEN function as o f C D K N IB alterations.. NKX3.I. Loss o f 8p appears to be an early event in prostate cancer and the most prom ising. candidate tum our suppressor gene at this site is NKX3.1. N KX 3.I encodes a prostate. specific homeobox gene that is likely to be essential for normal prostate developm ent.. NKX3.1 binds DNA and represses expression o f the PSA gene.[63] Loss o f NKX3.1. expression does appear to be related to the progression o f prostate cancer. One study. found that NKX3.1 was absent in 20 % o f lesions o f prostatic intraepithelial neoplasia,. 6% o f low stage prostate cancers, 22% o f high stage prostate cancers, 34% o f androgen. independent prostate cancers and 78% o f prostate cancer m etastases.[42] The loss o f. 19. this gene is o f particular interest as when normally present it represses expression o f the. PSA gene and the loss o f N K X 3 .1 m ay be involved in increased levels o f PSA with. prostate cancer progression.. KLF6. Kruppel-like factors (KLFs) are a group o f transcription factors that appear to be. involved in different biological processes including carcinogenesis. Significant genetic. alterations o f KLF6 have been reported including deletions and loss o f expression in a. minority o f high-grade prostate cancers.[64][65]. KLF6 and NKX3.1 have not been reported as frequently as the tum our suppressor. genes previously discussed and have been identified due to the fact that they are within. areas o f frequent allelic loss in prostate tumours.. Retinoblastom a (Rb). Retinoblastoma (Rb) has been reported as an important tum our suppressor gene in. many human cancers and prostate cancer is no exception. The disruption o f the normal. retinoblastom a regulatory pathw ay is associated with the pathogenesis o f many human. cancers. The retinoblastoma gene plays an important role in the G | phase o f the cell. cycle. It binds tightly to the E 2F fam ily o f transcription factors. When phosphorylated,. the Rb protein releases the E2F proteins causing transcriptional activation o f a variety. o f genes involved in cell grow th.[45] [66][67] Inactivation o f Rb appears to be. im portant in neoplastic transform ation, because expression o f w ild type Rb in Rb-. negative prostate cancer lines results in loss o f tum ourigenicity.[68] The predominant. 20. mechanism o f Rb inactivation involves allelic loss or m utation but decreased. transcription o f Rb has also been reported.[69][70]. p53. M utations in p53 are com m on in human neoplasms, but in prostate cancer it occurs. with a low frequency o f mutation. However p53 has an im portant role in prostate. cancer progression as abnorm al p53 expression is associated with bone metastases and. the developm ent o f androgen independent disease. A bnorm al p53 expression correlates. with high histological grade, high stage and clinical disease progression.. The p53 tum our suppressor gene product restricts entry into the synthetic phase o f the. cell cycle and promotes apoptosis in cells that are disorganised or have damaged DNA.. Loss o f normal p53 function results in uncontrolled cell growth.[71]. The analysis o f p53 expression can be ditTicult. The m utated p53 gene product has a. longer h alf life thus rendering it detectable by im m unohistochem istry. However. sensitive im m unohistochem ical techniques may detect over-expressed normal p53. It is. therefore more reliable to detect mutations in p53 by m olecular techniques. Abnormal. p53 expression is correlated with reduced survival after radical prostatectom y.[72]. 1.5.2 Oncogenes. c-myc and bcl-2 are well know n and im portant oncogenes not only in prostate cancer. but in many human cancers.. 21. c-myc. Several studies have dem onstrated increased myc expression in prostate cancer and a. significant correlation o f myc overexpression with Gleason grade. The myc oncogenes. are members o f the basic helix-loop-helix-leucine zipper (bHLHZ) fam ily o f. transcription factors. Myc proteins act as transcriptional activators or repressors through. dim erization or with other bH LH Z fam ily m em bers.[43] [70] [73][74][75]. bcl-2. The bcl-2 family o f genes are com m only expressed in primary and metastatic prostate. cancers, bcl-2 is not expressed in the normal prostate. Proteins expressed by the bcl-2. family have a crucial role in the regulation o f apotosis and the bcl-2 gene inhibits. program m ed cell death.[76][77][78] bcl-2 has also been im plicated in the development. o f androgen-independent prostate cancer due to its’ high levels o f expression in. androgen-independent cancers in the advanced stages o f disease.[79][80]. O ther oncogenes have only recently been recognized but may well em erge as novel. targets for molecular genetic intervention or through modified expression may allow. accurate prediction o f the m anner in which a neoplasm is likely to progress.. c-K it /Tyrosine Kinase Receptor. c-K it (tr-Kit) is a strong activator o f the Src-family tyrosine kinases. In a recent study it. has been reported that human tr-K it m RN A and protein are expressed in prostatic. 22. c a n c e r cells. It also describes fo r th e first tim e the e x isten ce o f a truncated c-K it protein. in p rim a ry tum ours and show a c o rre la tio n betw een tr-K it e x p re ssio n and activation o f. the Src pathw ay in the adv an ced stag es o f the disease. [81]. Stat5. A signal transducer and activ ato r o f transcription 5 (Stat5) has been identified as a. c ritical survival factor for p ro sta te c a n c e r c e lls .[82] A ctiv atio n o f Stat5 is also. a sso c ia te d w ith high histological g rad e o f prostate cancer.[83]. 1.5.3 Telomerase and Telomere Shortening. T e lo m e re length has been found to be strikingly sh o rter in p ro state c a n ce r including. p ro static intraepithelial n eo p lasia (P IN ) than in norm al prostate. T e lo m e re s stabilize. and p ro te c t the ends o f ch ro m o so m e s, but shorten b ecause o f cell d ivision and/or. o x id a tiv e dam age. C ritically short telom eres, in the se ttin g o f a b ro g ated D N A dam age. ch eck p o in ts, have been show n to c a u se chro m o so m al in sta b ility lea d in g to an increase. in c a n c e r incidence as a result o f ch ro m o so m e fusions, su b seq u e n t break ag e and re­. a rran g em en t. In norm al cells, su c ce ssiv e cycles o f c e llu la r rep lica tio n result in. p ro g re ssiv e loss o f telom eric seq u en ces. N orm al cells sense v ery sh o rt telom eres as. e v id e n c e o f D N A dam age and e x it the cell c y c le .[84] T e lo m e ra se is an enzym e that. m ain ta in s telom ere length by a d d in g rep etitiv e telo m eric seq u en ces to chrom osom e. ends. T e lo m erase activity is p re se n t in the m ajority o f p ro sta te c a n ce rs and not in. norm al p rostate e p ithelium .[85][86]. 23. 1.5.4 Androgen Receptor (AR). The androgen receptor has a critical role in prostate cancer. Androgen receptor. blockade can delay the progression o f prostate cancer and is used to treat patients. unsuitable for radical surgery or with cancer that has spread beyond the prostate. It has. been extensively studied in prostate cancer due to the fact that androgens are required. for the developm ent o f both the normal prostate and prostate cancer. Initially the. majority o f prostate cancers are sensitive to androgen deprivation.. H owever in patients with advanced disease, m ost tumours progress to an androgen. independent state with proliferation o f cells that do not require androgens for growth.. The mechanism o f acquired androgen insensitivity is unknown and has been the subject. o f much research, as androgen insensitive prostate cancers can no longer be treated. with endocrine therapy.. Mutations, am plifications and deletions o f the androgen receptor itself and structural. change in the androgen receptor protein, have been postulated as causing androgen. insensitivity.[47] [87][88][89][90][91]. In an analysis o f 44 mutant androgen receptors from prostate cancers, 16 per cent had. loss o f function, 7 per cent maintained w ild type function, 32 per cent dem onstrated. partial function and 45 per cent displayed a gain in function.[92]. Structural change o f the androgen receptor how ever has only been identified in a. minority o f androgen insensitive prostate cancers so therefore other factors must also be. involved in this phenomenon.. 24. G row th fac to r stim ulation m ay sensitise the androgen recep to r tra n sc rip tio n a l com plex. to sub physiological levels o f a n d ro g e n .[93] W e w ill refer to this topic o n c e again when. w e discuss the role o f grow th factors in p ro sta te cancer.. 1.7 Invasion and Metastasis Suppression Genes. For c a n c e r cells to spread to d istan t sites they m ust invade the strom a, p e n e trate the. v ascu latu re and im plant at d istant sites, and be able to survive there. C h a n g e s o f. a dhesion to the substratum are crucial fo r tu m o u r cell invasion and d ista n t m etastasis.. Several genes e ncoding proteins in volved in invasion and m etastasis in p ro state cancer. have been identified.. E -cadherins. T he cad h erin s are m em brane gly co p ro tein s that play an im portant role in c e llu la r. d ifferen tiatio n by m ediating cell-cell rec o g n itio n a n d adhesion. R e d u c tio n o f e-cadherin. ex p ressio n occurs is a com m on o ccu rren ce in prostate cancer, and has been reported to. co rrelate w ith tu m o u r grade, stage and s u rv iv a l.[46] [94][95][96] H o w e v e r the degree. o f E -c ad h e rin expression in prostate c a n c e r rem a in s controversial. N o rm al expression. o f e-c a d h e rin w as found in m ost p rostate c a rc in o m a cases exam ined in an. im m u n o h isto ch em ical study w hich sy ste m atic a lly ev alu ated E -c ad h e rin e x p re ssio n in a. broad ran g e o f form alin fixed prostate tis s u e .[97]. 25. Integrins. Normal basal epithelial cells in the human prostate express integrins however the. expression is abnormal or absent in the majority o f prostate cancer.[98] [99] [100]. C-CAM. C-CAM is expressed on the surface o f normal prostate epithelium how ever is absent in. most prostate cancers.[101] Loss o f C -C A M l expression occurs early in the. development o f prostate cancer, suggesting that C -C A M l may help maintain the. differentiated state o f the prostate epithelium. R e-introduction o f C -C A M l into cancer. cells can reverse their cancerous grow th.[102]. KAI1/CD82. M etastasis suppressor genes are defined as genes that do not affect cell growth o f. primary tumour cells but can inhibit development o f distant metastases.[103] C ancer. metastasis suppressor KAI1/CD82 belongs to the tetraspanin superfamily and inversely. correlates with the m etastatic potential o f a variety o f cancers including prostate. cancers. CD82 expression is reduced or absent in m ost prim ary prostate cancers and in. more than 90% o f m etastatic prostate cancers.[104][105] It is thought that the. mechanism o f K A Il/C D 82-m ediated metastasis suppression involves a cell-surface. protein physically associated with KAI1/CD82 nam ed KASP.[106]. 26. C D 44. C D 44 is ano tiier m etastasis su p p resso r gene for p rostatic can cer and C D 4 4 expression. is in v ersely co rrelated w ith histological grade, ploidy, and d istant. m etastases.[1 0 7 ][1 0 8 ] A dditional can d id ate m etastasis su p p resso r g en es h a v e been. identified fo r prostate cancer - N M E 23, m apsin, B R M SI, K ISSI and M A P2K 4.[103]. T he id en tifica tio n o f invasion and m etastasis suppression genes has p o ten tia l clinical. app licatio n s. P rostate cancers w ith loss o f these genes m ay rep resen t c a n ce rs w ith a. p o ten tially m etastatic phenotype. T hese cancers m ay require m ore a g g re ssiv e treatm ent. in c o n tra st to cancers, w hich have retain ed expression.. In o u r o p in io n the m ost p rom ising genes, w hich could be used as sp e cific targets for the. d etectio n , d iag n o sis and treatm ent o f p ro state cancer, include the tu m o u r su p p resso r. genes G S T P l , N K X 3 .1, PTEN and p27. N K X 3 .1, PT E N and p27 g e n e s also involve. grow th fac to r sign allin g pathw ays, w h ich hav e potential for m o le c u la r g e n etic. in terv en tio n . G enes, w hich play a role in d u cin g a n drogen stim u latio n o f th e prostate. gland such as A R , S R D 5A 2 and C Y P 17, are also potential targets fo r g e n e th erapy in. the future.. 27. CHAPTER 2. MATERIALS AND METHODS. 28. 2.1 Introduction. T his is an e x citin g era for m olecular path o lo g y as pow erful new investig ativ e to o ls are. being used now in research. C om paritive gen o m ic hybridisation allow s the scre e n in g o f. the entire genom e fo r chrom osom al aberrations by com p arin g flu o rescen ce ratio s for. norm al and tu m o u r D N A . C G H m icroarrays are a m odification o f this tech n iq u e w here. genes are spotted o n to a m icroarray as in F ig u re 1. T u m o u r D N A and norm al referen ce. DN A are h y b rid ised to the m icroarray and gene am plifications can be d e te c te d by. com p arin g flu o re sc e n c e ratios for norm al and tu m o u r DN A.. C G H m icro array tech n o lo g y was used in this w o rk in conjunction w ith lase r cap tu re. m icrodissection (L C M ) w hich facilitates the investig atio n o f a pure epithelial. population. A s the tech n iq u e o f laser capture m icro d issectio n is e x tre m e ly se le c tiv e the. q uantity o f D N A is sm a lle r using this technique th erefo re. D O P-PC R w as used in conjunction w ith LC M to am p lify the qu an tity o f D N A o b tain e d . from sam ples.. Low stringency P C R c o n d itio n s allow random p rim in g o f D O P p rim e r th ro u g h o u t the. g enom e w hich resu lts in am plification o f the w h o le genom e as o p p o se d to a specific. target.. T hese tech n iq u es w ere used to identify genetic a m p lificatio n s in o u r p ro sta te sam p les. and are ou tlin ed in m o re detail below (2.1-2.3).. A m p lification o f T o p o iso m e rase II alpha gene w as identified in o u r p ro sta te sam ples. u sin g the C G H m ic ro arra y technology.. Im m u n o h isto ch em istry w as used to investigate w h e th e r increased ex p re ssio n o f . T o p o iso m erase II a lp h a gene w as present in a larg e r co h o rt o f prostate c a n c e r and. 29. benign prostatic hyperplasia cases and the details o f how this was carried out is also. outlined below (2.4-2.5).. 2.1 CGH Microarray A nalysis. 2.1.1 Samples:. W e analysed 4 prostate samples using the Vysis GenoSensor. 2 o f the prostate samples. were from autopsy cases o f advanced prostate cancer. One o f these cases (Gleason. Score 9) had been treated with anti-androgens and was known to be hormone resistant.. The second case was a case o f m etastatic prostate cancer which had not been treated by. anti-androgens. The third prostate sample was a TURP surgical specim en with. involvem ent o f prostate chippings by Gleason Score 7 prostate adenocarcinom a. The. fourth sam ple was a TURP surgical specim en with benign prostatic hyperplasia (BPH). and no evidence o f prostatic carcinoma.. 30. 2.1.2 Laser capture microdissection:. Laser capture microdissection (LCM) permits the rapid and rehable procurement o f pure. populations of cells from tissue sections, in one step, under direct microscopic visualization. while maintaining nucleic acid and protein content. LCM is a powerful upstream tool. facilitating the generation o f informative data from downstream technologies such as CGH. arrays, cDNA arrays and Taqman PCR.. Laser capture microdissection was performed on each of the cases as previously. described.[109] Briefly a laser microdissection system (Arcturus Pix Cell, Arcturus. Engineering) was used to isolate prostate epithelial cells from each specimen according. to the manufacturer’s instructions. First the laser was used to remove the surrounding. stromal cells from an area o f interest. Then the target cells were attached to the cap by. Firing the laser. The cap was transferred to a sterile microcentrifuge tube containing. 40|il proteinase K extraction buffer ( lOmM. Tris HCL, pH 8.0, ImM EDTA, proteinase K 40|il/m l, 1% Tween 20, pH 8). The tubes. were inverted at 55°C overnight. The supernatant containing the DNA (approx. 350|ll. from laser microdissected tissue) leaving behind the precipitated protein pellet was. poured into a clean 1.5ml microfuge tube which contained 300(ll 100% Isopropanol. and 35|ll 3M sodium acetate. As the DNA yield was expected to be low due to the use. of microdissected tissue we added lfj.1 of glycogen solution as a DNA carrier. The. sample was mixed by inverting gently 50 times. It was then centrifuged at 16,000xg for. 5 minutes. The supernatant was poured off and the tube was drained on clean absorbent. 31. paper. 300|ll 70% ethanol was added and the tube was inverted to wash the DNA pellet.. The sample was then centrifuged at 16,000xg for 1 minute. The ethanol was carefully. poured off. The tube was inverted and drained on clean absorbent paper and allowed to. air dry for 10-15 minutes and dissolved in 6)J.l o f water. The sam ple was incubated. overnight at room temperature. The DNA was then stored at 4°C.. Figure 1: Example o f laser capture microdissection on benign prostate tissue. B E F O R E A F T E R C A P T U R E D M A T E R IA L. 32. Figure 2: Example o f laser capture microdissection on prostate cancer. B efore m ic ro d issec tio n. A fter m ic ro d issec tio n. 2.2 DO P-PCR and Nick Translation. Figure 3: Diagramatic representation o f DOP-PCR. DS Genomic DNA. Low stringency PCR conditions allow random priming of DOP primer throughout the genome. DOP-PCR allo^vs Random Amplification of Multiple loci and amplification o f the entire genome. 34. Several authors have reported the feasibility o f com bining laser capture microdissection. with DOP-PCR and C G H .[110] The D O P-PCR was performed using the DOP-PCR. m aster kit (B oehringer Mannheim, M annheim , G ermany) according to the. m anufacturer’s instructions. Ing o f male reference DNA and Cosh DNA also under 2. rounds o f D OP-PCR amplification. Briefly in a total volume o f 50|il using 5(0.1 o f test. DNA tem plate or 5|0.1 o f reference containing Ing o f male reference DNA the sample. was heated to 94°C followed by 5 cycles o f 94°C for 1 minute; 30°C for 1.5 minutes;. and a ramp o f 30°C-72°C at 23°C/second for 3 minutes. Then 25 cycles followed at. 94°C for 1 minute; 62°C for 1 minute and 72°C for 3 minutes +10 seconds per cycle.. A fter a final extension period o f 10 minutes at 72°C, the sample was cooled to 4°C.. 5(il o f the DOP-PCR product was used as a tem plate for the 2"‘* round DOP-PCR. reaction, which was performed at tem perature, and time settings identical to the P '. round DOP-PCR. 10|ll o f the 2"̂ * round DOP-PCR product were used to check the. fragm ent size on a 2% agarose ethidium brom ide gel. Samples were taken into analysis. if a sm ear could be detected after the second round o f DOP-PCR. DNA labelling was. performed by nick translation. 10|ll o f the D O P-PC R product was used as a template. for the reaction. The reaction was perform ed in a total volum e o f 5 0 |ll with the. follow ing reagents: 2(J,1 o f ImM A lexa-488 (G reen), 17|ll o f dNTP mix, 5|ll lOX nick. translation buffer, 10|Xl nick enzym e mix and 6 |il o f water. A sim ilar reaction using. 10|il o f the DOP-PCR product o f Ing o f male reference DNA and C osh DNA which. had undergone 2 rounds o f DOP-PCR am plification and 2|ll o f Im M A lexa-594 (Red). was used instead o f 2|0.1 o f ImM Alexa-488 (G reen) for reference cases.. 35. 2]i\ o f the nicked DNA (tests and references) were used to check the fragm ent sizes on. a 2% agarose ethidium bromide gel.. Figure 4: 2" ̂ round DOP-PCR products o f the 4 prostate samples (sam ple 1-4).. Sample 5 represents a positive control as does the lane im m ediately to it’s right. DNA ran below the 300 nucleotide m arker with tails extending up to about the. 600 nucleotide marker.A negative control is present in the extrem e right hand. lane.. 36. 2.3 CGH DN A Microarray. Figure 5. C om parative Genomic Hybridization (CGH) to Chromosomes and A rrays. Test Genomic DNA Reference Genomic DNA. C o t - 1 DNA. o o o • o o o o. ■. (0 cr. Position on Chromosome. - w V *I. Position on S equence. We applied a com m ercially available genomic D NA m icroaray kit (Vysis G enosensor). and hybridised the nicked products from our 4 prostate samples and 4 references to. Amplionc 1 D NA microarrays containing 57 oncogenes as per instructions supplied by. the m anufacturer. Briefly the labelled DNA (4|il o f green test and 4 |il red reference. were m ixed with 22|ll microarray hybridisation buffer (Vysis) containing Cot-1. followed by incubation at 80° for 10 minutes to denature the DNA. 30|J.l o f. hybridisation m ixture containing denatured DNA was dropped on the chip slide. following incubation o f the m icroarray at 37° for at least 18 hours in an incubator. A fter. hybridisation the slides were washed in 3 series o f w ashing solutions (50%. 37. fo rm am id e/2 X S S C ) w ith incubations at 40° (10 m inutes each), and 4 series o f w ashing. s o lu tio n s ( IX SSC ) at room tem perature and c o u n terstain ed w ith 18|il 4 ’6-. diam id in o p h en y lin d o le (D A P I) IV solution. (V ysis).. T h e h y bridised m icroarray slides w ere analysed w ith m ic ro arra y read er and analysis. so ftw a re (G enosensor A rray 300). G reen (test) to red (referen ce) (G /R ) ratios w ere. a u to m a tic ally determ ined for each sam ple and the norm alised G /R ratio w as taken to. rep re sen t the relative av erag e nu m b er o f copies o f the seq u en ce fo r those spots that. w ere selected as controls. S pots w ith G /R ratios m ore than the m ean plus three standard. d ev iatio n s ( -1 .3 ) w ere c o n sid e red as am plifications o r g ain s o f the indicated copy. num ber.. A control experim ent w as run in tandem w ith the e x p erim en t u sin g cell line DN A . (C osh) m ixture com prising 3 cell lines) provided by the m an u fa ctu rer w ith know n. d o c u m e n ted am plifications o f a variety o f oncogenes. O u r resu lts w ere exactly. c o n c o rd a n t with those d e scrib ed by Vysis.. 38. Figure 6: Hybridisation o f tum our and reference DNA. Figure 7: Summary o f m ethods utilised to obtain report on Fluorescence. LaMr captur* microdtn«ction of tiSMM sanpi* DNA •xtracUon. DOP^CR amplM cationMicroarray. DNAI#>«llng. Preparation Hybridlaatlon. Imaqa cip tu rt. Imaga anaiyila. 2.4 Evaluation o f expression o f Topoisomerase gene (amplified on CGH. microarrays) by immunohistochemistry. Imm unohistochem istry was used to evaluate the expression o f one o f the genes. (Topoisom erase II alpha gene) in w hich amplifications were detected on CGH microarrays.. Expression o f this gene was evaluated in a larger cohort o f prostate cancer and benign prostatic. hyperplasia cases.. 2.4.1 Patients and tumours used in immunohistochemical study. The im munohistochemical study included 100 patients with adenocarcinom a o f the. prostate and 42 patients with benign prostatic hyperplasia (BPH). T he adenocarcinom a. cases included 59 transurethral resection o f prostate (TURP) specim ens and 41 radical. prostatectom y specimens. The 42 BPH cases were transurethral resection o f prostate. (TURP) specimens. These cases were diagnosed during the period 1999-2003 and were. identified by performing a SNO M ED code search o f the Adelaide & M eath Hospital. Incorporating the National C hildren’s Hospital Cellular Pathology database. Formalin. fixed blocks from these cases w ere retrieved from the files. Tissue m icroarrays were. constructed as described previously.[l 11] 138 cases were present in triplicate and. included representative areas o f tum our o r glandular tissue in cases o f BPH. Sufficient. tissue from one case o f BPH was not represented on the tissue m icroarray and this case. was excluded. 3 cases o f adenocarcinom a were present in duplicate only as there was. insufficient tissue from the third core present on the microarray. Histopathological 40. reports which included clinical details were reviewed for all adenocarcinom a cases and. cases were classified into 4 categories benign prostatic hyperplasia (n=41), prostate. carcinom a Gleason Score 6 (n=28), prostate carcinoma G leason Score 7-8 (n=35), and. prostate carcinom a Gleason Score 9-10 (n=37). We also com pared prostate carcinomas. G leason Score 8-10 under the categories o f hormone resistant (n=14) and hormone. responsive (n=14). The 14 cases categorised as hormone resistant were recurrent. prostate adenocarcinom as present in transurethral resection o f prostate (TURP). specim ens when clinical details indicated that the patient had received anti-androgen. treatm ent. These cases were G leason Score 8-10. 14 cases o f m atched Gleason Score. prostate carcinom as who had not received anti-androgen treatm ent w ere com pared with. these cases using the U Mann W hitney test.. 2 .4 .2 Im m unohistochem ical staining. Im m unohistochem istry for Topoisom erase II-Alpha was perform ed using a monoclonal. anti- Topoisom erase II-Alpha antibody with a dilution o f 1:300. Sections o f 5|im . thickness were studied. Antigen retrieval was performed with dew axed sections using a. pressure cooker and the Trilogy system for 10 minutes. A standard avidin-biotin-. peroxidase com plex technique (D AK O ) was used for visualisation w ith. diam inobenzidine as a chromogen. Sections were counterstained w ith haem atoxylin. and mounted. Tonsil samples from our routine files were used as positive controls for. Topoisom erase II-Alpha.. 41. 2.4.3 Evaluation o f Immunohistochemistry. Im m u n o h isto ch em ical staining w as ev a lu a te d light m icroscopically u sing an X 40. o bjective a n d a M ille r ocular. T he M iller o c u la r eyepiece gave a square field, in the. c o m e r o f w h ich w as a sm aller ruled square, o n e -n in th the area o f the total square.. N uclei w ith stro n g hom ogenous T o p o iso m e rase II-A lpha staining w ere c o u n te d in the. large square a n d the total num ber o f nuclei, b o th p o sitiv e and negative sta in in g counted. in the sm all sq u are. T he num ber o f cells in the sm all square w as m u ltiplied by 9 to. obtain the total n u m b er o f cells in the large square. A m ean o f 240.5 ep ith elial cells. w ere c o u n ted fo r the prostate cancer cases a n d a m ean o f 88 epithelial cells co u n te d for. the cases o f b e n ig n prostatic hyperplasia. 5 n o n -o v erlap p in g square fields w ere counted. for each tissu e co re, 3 in the y-axis and 2 in th e x-axis. T he percentage o f p o sitiv ely . staining nuclei w ithin the 5 large squares w as c alcu lated for each core. In cases present. in triplicate an av e ra g e result w as obtain ed fro m 3 cores and in cases p resen t in. duplicate an av e ra g e was o btained from 2 cores. T his result was the T o p o iso m e rase II-. A lpha index fo r the case. T his m ethod has been used in previous studies in the. evaluation o f im m u n o h isto ch em ical results in tissue m icro array s.[ 1 1 2 ][1 13]. In addition ea ch T o p o iso m erase II-A lpha in d ex w as o btained by im age an aly sis using. the A riol system fo r tissue m icroarrays. A n a ccep tab le level o f co n c o rd a n c e w as taken. as w ithin 10% o f T I index obtained u sin g the M iller ocular.. O f 140 c ases in total (138 present in triplicate and 2 present in duplicate) 14 cases. (10% ) sh o w ed d isc o rd a n ce b etw een A riol im ag e an a ly se r and the M iller o c u la r TI. indices. In all c a se s the A riol im age an a ly se r o v e re stim ate d TI indices c o m p a red to the. M iller o c u la r m ethod. 42. A second observer reviewed these discordant cases. This discordant group was. com posed o f one G leason score 7, two G leason score 8, nine Gleason score 9, and two. Gleason score 10 cases. These 14 cases tended to have higher Topoisom erase indices. (mean TI o f 13%) using the M iller ocular than the average Topoisom erase index for all. 99 carcinom a cases analysed (mean TI o f 3.69%).. Review o f these discordant cases by a second observer using light m icroscopy and a. Miller ocular correlated within 10% o f the original Topoisom erase indices obtained by. the first observer. The discrepancy between the Ariol image analysis results and the. light m icroscopy results was due to a difficulty by the image analyser in distinguishing. Topoisom erase negative tumour cells from strom a when there was infiltration o f the. stroma by less differentiated tumour. It therefore under estimated the num ber o f total. tumour cells within a field o f analysis hence leading to an over-estimation o f % cells. positive for the Topoisom erase II alpha antibody.. Examples o f im m unohistochem ical staining results obtained are presented in Figures 8. and 9.. 43. A C E. • V j f v . vV.» y - m r h -. • V v V - V . -. I• / ^• t* . •*- h. B '. • • •. ♦ » •. •. %. •. « \. D 1 i *. %. I. f , ^ m. • - • 1 * 1 * *. ‘ 1. Figure 8 A-F: Im m unohistochem ical Staining for T O PO II-A lp h a in. (A) Low pow er view o f T M A show ing T O PO II-A lpha focal positivity in benign prostatic. h yperplasia (BPH).. (B) High pow er view o f a benign prostatic gland show ing focal T O PO II-A lpha nuclear positivity.. (C) Low pow er view o f T M A o f a prostatic ad en o carcin o m a (G leason score 3-1-3) show ing focal. cell positivity for T O P O II-Alpha.. (D) High pow er view o f prostatic adenocarcinom a (G leason score 3+3) show ing focal discrete. nuclear T O P O II-A lpha positivity in prostatic acini.. (E) Low pow er view o f T M A o f prostatic adenocarcinom a (G leason score 9) show ing m ultiple. cells positive for T O P O II-Alpha.. (F) High pow er view o f prostatic adenocarcinom a (G leason score 9) show ing discrete n u clear. T O PO II-A lpha positivity in m any tum our cells w ithin the lesion.. 44. J* , •. >f '-̂ i? • * •• “> s. • - » • • , , ^. »» %7 y .. Figure 9: H ig h p o w e r v iew o f a n d ro g e n in d e p e n d e n t p ro s ta tic a d e n o c a rc in o m a (G le a s o n sc o re 9 ) sh o w in g d is c re te n u c le a r T O P O II-A lp h a p o sitiv ity in m a n y tu m o u r ce lls w ith in th e le sio n .. 45. 2 .5 Statistics:. Data were entered on to a com puterised database and analysed by the SPSS statistical. package using the U Mann W hitney test.. 46. CHAPTER THREE. RESULTS OF CGH MICROARRAYS. 47. Benign prostatic hyperplasia. Figure 1 0: M icroarrays Showing fluorescence em issions. Localised prostate cancer. CGH MICROARRAYS. Androgen independent prostate cancer. M etastatic prostatic cancer to bone marrow. 48. 3.1 ARRAY CGH ANALYSIS. Figure 11: CGH M icroarray A m pliOnc G enoSensor software. A report is generated for each array. This report tabulates among other variables the. assigned target number, target clone nam e, cytogenetic location and the norm alised test. to reference intensity and a pooled correlation co-efficient. N orm alised test to reference. intensities more than the mean plus three standard deviations (-1 .3 ) w ere considered as. am plications or gains o f the indicated copy number.. One o f these cases (Gleason Score 9) had been treated with anti-androgens and was. known to be horm one resistant. The second case w as a case o f m etastatic prostate. cancer w hich had not been treated by anti-androgens. The third prostate sam ple was a. 49. TURP surgical specim en with involvement o f prostate chippings by Gleason Score 7. prostate adenocarcinom a. The fourth sample was a TU R P surgical specimen with. benign prostatic hyperplasia (BPH) and no evidence o f prostatic carcinoma.. Figure 12: Exam ple o f report o f fluorescence ratios generated by A m pliO nc GenoSensor software. A m p l i O n c ™ I. V i '. 1 1 1 1 ■ I ■ I I ■ 1 1 1 1 1 1 1 I 1 1 1 ■ 1 1 1 1 1 1 P r o b e. 50. T able 5:. A m plifications o f the follow ing genes w ere id en tified fo r the case o f androgen. independent adv an ced prostate cancer:. G ene N orm alised test to refe re n c e . intensity (flu o re sc e n ce . ratio). Pooled c orrelation c o ­. efficient. M ET. (tyrosine kinase, am plified. in the transition betw een. p rim ary tum ours and. m etastases). 4.27 0.91. H RAS. (Plays role in norm al. grow th and d ifferen tiatio n ). 4.11 0.85. C B FA 2. (core bind in g fac to r runt. d om ain, involved in A M L ). 3.96 0.93. TOP2A. (Involved in cell. replication). 3.63 0.85. FES. (feline sarcom a virus). 2.96 0.91. 51. T able 6:. A m plifications o f the fo llo w in g genes w ere id en tified fo r the case o f m etastatic prostate. cancer:. G ene N orm alised test to reference. intensity (fluorescence. ratio). P ooled correlation c o ­. efficient. FES. (feline sarcom a virus,). 3.42 0.86. M YB. (A lterations in M Y B gene. in m ore than 1/3 o f hum an. solid tum ours). 2.19 0.87. B C L 2 3 ’. (B locks the apoptotic death. o f cells ie. lym phoctes). 1.85 0.91. TOP2A. (Involved in cell. replication). 1.74 0.91. A R 5 ’. (C an facilitate tum our cell. g row th in low androgen. concentrations. 1.70 0.87. 52. T able 7:. A m plifications o f the following genes were identified for the case o f Gleason score 7. localised prostate cancer:. G ene Normalised test to reference. intensity (fluorescence. ratio). Pooled correlation co­. efficient. M ET. (tyrosine kinase,). 5.36 0.82. SA S/CD K . (Sarcom a amplified. sequence,). 3.17 0.83. PG Y l. (P-glycoprotein l,a ls o . know n as multi-drug. resistance 1). 2.61 0.87. TOP2A. (Involved in cell. replication). 2.46 0.88. ER B 2. (oncogene, most com monly. am plified in breast. carcinom a). 2.24 0.85. 53. Table 8:. A m plifications o f the following genes were identified for the case o f benign prostatic. hyperplasia:. Gene N orm alised test to reference. intensity (fluorescence. ratio). Pooled correlation co­. efficient. FES. (feline sarcom a virus). 4.04 0.92. PIK3CA. (Phosphatidylinositol, 3-. kinase, sorts proteins to the. vacuole). 3.56 0.88. GLI. (Encodes a protein product. with 5 zinc finger DNA. binding motifs). 2.89 0.96. MOS. (M oloney murine sarcom. viral oncogene homolog,). 2.06 0.94. TOP2A. (Involved in cell. replication). 1.54 0.97. 54. As can be seen from the above tables am plification o f M ET gene w as identified in the. case o f localised prostate cancer.. FES gene was amplified in androgen independent advanced prostate cancer, metastatic. prostate cancer and benign prostatic hyperplasia.. H ow ever increased copy number o f Topoisom erase II-Alpha (T 0 P 0 2 A ) was detected. in all 4 prostate samples including the sample o f BPH. A dvanced horm one insensitive. prostate carcinom a showed the most marked increase in copy number.. The results for the target o f interest Topoisom erase II-Alpha are presented in Table 9. Table 9: A rray CGH analysis for Topoisom erase II-Alpha gene. N orm alised test to. reference intensity. (fluorescence ratio). Pooled correlation co­. efficient. A dvanced hormone. insensitive prostate. carcinom a. 3.63 0.85. M etastatic prostate. carcinom a. 1.74 0.91. Localised G leason Score 7. prostate carcinom a. 2.46 0.88. 55. T op o iso m erase II-A lp h a is an essential c e llu la r enzym e that functions in the. segregation o f n e w ly replicated ch ro m o so m e pairs, in chrom osom e c o n d e n sa tio n and in. altering D N A superhelicity.. As T o p o iso m e rase II-A lpha gene w as sig n ific a n tly am plified in all o u r cases o f prostate. cancer and as it has been reported as a p ro g n o stic m arker for tissues o th e r than prostate. this gene w a rra n te d fu rth e r investigation as reg a rd s to it’s expression in p rostate cancer.. T he fact that c h em o th erap eu tic drugs that targ et the T o p oisom erase II a lp h a gene are. currently a v a ilab le increased the im portance o f investigating its ’ ex p ressio n in prostate. tissue.. 56. CHAPTER FOUR. RESULTS OF IMMUNOHISTOCHEMICAL STUDY ON. TOPOISOMERASE II ALPHA GENE EXPRESSION IN BENIGN. PROSTATIC HYPERPLASIA AND PROSTATE CANCER. 57. 4.1 Introduction. As Topoisom erase II-Alpha gene was significantly am plified in all our cases o f prostate. cancer and as it has been reported as a prognostic m arker for tissues other than prostate. this gene warranted further investigation as regards to it’s expression in prostate cancer.. The fact that chem otherapeutic drugs that target the Topoisom erase II alpha gene are. currently available increased the importance o f investigating its’ expression in prostate. tissue.. Examples o f im m unohistochem ical staining for the Topoisom erase II-Alpha antibody. are presented in Figures 13 and 14.. 58. A c E ♦ ' *• '•. ’.' C • * • v \ •*. A. : ; * ~. ,•• • • • • i V - . •>.. • »• ' i . ••• ' ’•. 8 ' . r 1 *. •. % «. • «. % \. D. t yL. %. '«. %. f S * 1 ^ p m * . .. , ̂ r ^ .. * i f ’ * : ^ \ 9 k ' - ^ .. <• ' • * r \ n. _____ ________ A - L h _____. Figure 13 A-F : Im m unohistochem ical Staining for T O PO Il-A lp h a in. (A) Low pow er view o f T M A show ing T O PO II-A lpha focal positivity in benign prostatic. hyperplasia (BPH).. (B) High pow er view o f a benign prostatic gland show ing focal T O PO II-A lpha nuclear positivity.. (C) Low pow er view o f T M A o f a prostatic ad en o carcin o m a (G leason score 3+3) show ing focal. cell positivity for T O P O II-Alpha.. (D) High pow er view o f p ro static adenocarcinom a (G leason sco re 3+3) show ing focal d iscrete. nuclear T O PO II-A lpha positivity in prostatic acini.. (E) Low pow er view o f T M A o f prostatic adenocarcinom a (G leaso n score 9) show ing m ultiple. cells positive for T O P O II-Alpha.. (F) H igh pow er view o f p ro static adenocarcinom a (G leason sco re 9) show ing discrete nuclear. T O PO II-A lpha positivity in many tu m o u r cells w ithin th e lesion.. 59. I. I. i«i • • ' ■ f 1 % T • *. F igure 14: High p o w er view o f androgen independent prostatic adenocarcinom a (G leason sco re 9) show ing discrete nuclear T O P O II-A lpha positivity in m any tu m o u r cells w ithin the lesion.. 6 0. 4 .2 Statistics:. D ata were entered on to a com puterised database and analysed by the SPSS statistical. package using the U Mann W hitney test.. Analysis o f im munohistochemical results;. The U Mann Whitney test was used to determine w hether there w as a statistically. significant difference between the Topoisom erase II-Alpha indices in each group.. The median and range o f Topoisom erase II alpha indices (TI) for each Gleason score. are presented in Table 1 and median and range for the categories statistically analysed. presented in Table 2 and 3.. T here was no statistically significant difference between the groups benign prostatic. hyperplasia and prostate carcinom a G leason Score 6 (P=0.245). The difference between. the groups prostate carcinom a G leason Scores 6 and 7 was statistically significant. (?<0.008). In addition there w as a highly statistically significant difference between the. groups prostate carcinoma G leason Score 7 and Gleason scores 8,9 and 10 (not selected. for horm onal status), (P=0.000).. W hen the category o f G leason score 8,9,10 was analysed as regard to hormone. treatm ent and insensitivity (n=15) and cases with no hormone treatm ent (n=34) the. difference in Topoisom erase indices approached statistical significance (P=0.081).. R esults o f the U M ann-W hitney test statistical analysis are presented in Table 10-13.. Scatterplots were generated from the data. (Figures 15 & 16). 61. T able 10: Median and range o f Topoisom erase indices according to G leason score. Gleason. score 6. Gleason. score 7. Gleason. score 8. Gleason. score 9. Gleason. score 10. N um ber o f. cases. 28 22 12 29 8. Median 0.3 0.9 1.65 2.6 12.55. Range 0-3.6 0-5.9 0.3-8.8 0-54.8 0.7-48.6. Table 11: Median and range o f Topoisom erase indices according to categories. analysed. Benign. prostatic. hyperplasia. Gleason. score 6. Gleason. score 7. Gleason. score 8,9,10. Num ber o f. cases. 41 28 22 49. Median 0 0.7 0.9 15.1. Range 0-3.3 0-3.6 0-5.9 0-48.6. 62. Table 12: M edian and range of Topoisom erase indices according to categories. analysed. Gleason score 8,9,10,. hormone resistant, post. hormone deprivation. Gleason score 8,9,10, no. hormone treatment. N um ber o f cases 15 34. M edian 5.5 2.2. Range 0-48.6 0.3-36.3. 63. Table 13: Statistical analysis o f Topoisom erase II alpha indices. Categories analysed for statistical significance between. Topoisom erase II alpha indices. Z test. statistic. P value. Benign Prostatic H yperplasia and Prostate C arcinom a Gleason. Score 6. -1.163 0.245. Prostate Carcinom a G leason Score 6 and Prostate Carcinom a. Gleason Score 7. -2.636 0.008. Prostate Carcinom a G leason Score 7 and Prostate Carcinom a. Gleason Score 8,9,10. -3.601 0.000. Hormone resistant Prostate Carcinoma G leason Score 8-10 and. Gleason Score 8-10 with no hormone treatm ent. -1.747 0.081. 64. G leas o n sco re 6 G leas o n sco re 7 G leas o n sco re 8 ,9 ,1 0. B e n ig n prostatic h yp erplas ia. Diagnostic category. Figure 15: Scatterplot for Topoisomerase II-A lp h a indices as determ ined by diagnostic category. Diagnostic category O Gleason score 6 ^ Gleason score 7 y Gleason score ^ 8,9,10 ^ Benign prostatic. hyperplasia. 65. T op. oi so. m er. as e. in de. x O. O. Hormone treatment No hormone treatment. Diagnostic category. Figure 16: Scatterplot for Topoisomerase II-AIpha indices comparing hormone resistant prostate cancer with group of prostate cancer with no hormone treatm ent. D iagnostic category O H orm one treatm en t —I No horm one. ' tre a tm e n t. 66. CHAPTER FIVE. DISCUSSION. 67. 5.1 Introduction. Id en tificatio n o f features, w hich ac cu ra te ly predict the b e h a v io u r o f p ro sta te cancer. o c c u rrin g w ithin a specific p atient, is a m ajor challenge. G leaso n g ra d in g is based on. m orph o lo g ical features and is a p ow erful prognostic in d icato r h o w e v e r there can be. d ifficu lties w ith in terobserver reproducibility. In addition p ro state carcin o m as, w hich. are m o rp h o lo g ic a lly in d istin g u ish ab le and discovered incidentally, can behave in a. c lin ic a lly indo len t fashion or aggressively. T he identification o f g e n e s by the new m icro. array techn o lo g y , w hich correlate w ith patient prognosis, is an e x c itin g developm ent. w ith p o tential clinical application.. In this stu d y increased T o p o iso m e rase II-A lpha im m unoexpression w as associated with. p ro sta te c a n c e r w ith the highest ex p ressio n in horm one resistan t p ro sta te carcinom as. It. also co rre la ted w ith the know n p ro g n o stic m arker o f G leason Score.. A m p lific a tio n o f the T o p o iso m e rase II-A lpha gene was identified in the four prostate. sam ples investigated by C G H m ic ro arra y indicating that in creased e x p ressio n o f the. gen e is d u e to am plification at a D N A level.. 5.2 Topoisomerase II-Alpha gene. T o p o iso m e rase II-A lpha is an esse n tia l cellu lar enzym e that fu n ctio n s in the. seg re g a tio n o f new ly rep licated c h ro m o so m e pairs, in c h ro m o so m e c o n d e n sa tio n and in. a lterin g D N A superhelicity. D N A to p o iso m erases, are in volved in n early all biological 68. processes governing DNA and untangle intertwined DNA strands prior to cell division. by transiently breaking and then religating duplex strands o f DNA. Chem otherapeutic. drugs that target Topoisom erase II-Alpha such as etoposide, doxorubicin and. m itoxantrone act by stabilizing a norm ally transient D N A -topoisom erase II complex,. thus increasing the concentration o f double-stranded DNA breaks. This phenomenon. triggers mutagenic and cell death pathw ays.[1 14][115] Topoisom erase II-Alpha has. been reported as a prognostic m arker in a variety of. tissu es.[l 16][113][117][118][119][120][121][122][123][124][125][126][127]. To our knowledge there are only 2 papers in the literature which investigate. Topoisom erase Il-alpha expression in prostate cancer.[l28][129] These studies do not. investigate Topoisom erase II-Alpha expression in hormone resistant prostate. carcinom a. This is o f interest as Topoisom erase II-Alpha is the target o f the drug. etoposide that is an active agent in the com bined chem otherapy o f horm one-insensitive. prostate carcinom a in clinical trials.[130][131]. In this study we examine Topoisom erase II-Alpha expression in benign prostatic. hyperplasia and prostate cancer categorised by Gleason Score and by horm one. sensitivity and investigate w hether Topoisom erase II-Alpha expression is significantly. increased in different diagnostic categories o f prostate cancer.. There is little information regarding the expression o f Topoisom erase Il-alpha in. prostate carcinom a or in particular in identifying w hether its’ expression is increased in. com parison with benign prostatic hyperplasia. To our know ledge there is only one. paper in the literature which attempts to com pare Topoisom erase Il-alpha expression in. prostate cancer and benign prostatic hyperplasia.[129] This study reported little. 69. expression o f Topoisom erase II-Alpha in benign prostatic hyperplasia how ever it only. investigated 10 cases. In contrast to this study we identified Topoisom erase II-Alpha. expression in benign prostatic hyperplasia (mean Topoisom erase index 0.9). In addition. in our study there was no statistically significant difference in Topoisom erase II-Alpha. expression between Gleason Score 6 prostate carcinom a and benign prostatic. hyperplasia.. The previous tw o studies conclude that Topoisom erase II-Alpha expression correlates. with Gleason Score. Our larger study also supports this finding. We also identified a. statistically significant difference in Topoisom erase II-Alpha expression between. prostate cancers classified as regards to Gleason Score.. Am plification o f the Topoisom erase II-Alpha gene was identified in the four prostate. samples investigated by CGH microarray indicating that increased expression o f the. gene is due to am plification at a DNA level. The degree o f am plification w as greatest in. the hormone resistant prostate carcinom a how ever amplification o f the gene was also. identified in the case o f benign prostatic hyperplasia. As Topoisom erase II-Alpha gene. is a proliferation related gene and benign prostatic hyperplasia is a proliferative. condition this is not surprising. This also correlates with the fact that we did not. identify a statistically significant difference in Topoisom erase II-Alpha expression. between G leason Score 6 prostate carcinom a and benign prostatic hyperplasia.. Our study is the first study to our know ledge to examine Topoisom erase II-Alpha. expression in horm one resistant prostate carcinom a. This is o f particular interest as. Topoisom erase II-Alpha is the target o f the drug etoposide, which is an active agent in. the com bined chem otherapy o f horm one-insensitive prostate carcinom a in clinical. trials. We identified increased Topoisom erase II-Alpha expression in the hormone. 70. resistant prostate c a rc in o m a s. T his correlates w ith the fac t that the clinical su b g ro u p o f . horm one resistan t p ro sta te carcinom as has show n the b e st response to. etoposid e.[1 3 0 ][1 3 1 ] A p o tential clinical ap p lication o f the evaluation o f. T opoisom erase II-A lp h a e x p ressio n in prostate c a n ce r tissu e could be used to design. rational c o m b in atio n th erap ies w ith T opoisom erase II-A lp h a targeting drugs.. High T o p o iso m e rase II-A lp h a im m unoexpression w as asso ciated w ith h orm one. resistant prostate c a rc in o m a s. It also correlated w ith the know n prognostic m ark er o f. G leason Score. A n u m b e r o f studies have co rre la ted the level o f T o p o iso m erase II-. A lpha ex p ressio n w ith a resp o n se to an ti-T o p o iso m erase II-A lpha drugs in c a n c e r cell. lin es.[132][133][134] In the future evaluation o f T o p o iso m e rase II-A lpha e x p re ssio n in. prostate can cer tissu e c o u ld be used to design rational com b in atio n therapies w ith. T opoisom erase II-A lp h a targ etin
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