MICRO RNA-21 EXPRESSION LEVELS IN INVASIVE BREAST CARCINOMA WITH A NON-INVASIVE COMPONENT

Arch. Biol. Sci., Belgrde, 67(4), 1285-1295, 2015 DOI:10.2298/ABS150327105P MICRO RNA-21 EXPRESSION LEVELS IN INVASIVE BREAST CARCINOMA WITH A NON-INVASIVE COMPONENT Nin Petrović 1,*, Snežn Jovnović-Ćupić 1, Gorn Brjušković 2, Silvn Lukić 3, Jelen Rognović 4, Milen Krjnović 1 nd Vesn Mndušić 1 1 University of Belgrde-Vinč Institute of Nucler Sciences, Belgrde, Serbi 2 Fculty of Biology, University of Belgrde, Belgrde, Serbi 3 Institute for Oncology nd Rdiology of Serbi, Belgrde, Serbi 4 Deprtment of Phrmcology, Fculty of Dentistry, University of Belgrde, Belgrde, Serbi *Corresponding uthor: drgoninspirtion@yhoo.com Abstrct: Invsive ductl crcinoms with non-invsive component (IDC-DCIS) re clssified s group of invsive brest crcinoms, together with pure invsive ductl crcinoms of the brest (IDC). MicroRNA-21 (mir-21) hs been chrcterized s fctor of brest cncer invsiveness, however the difference in mir-21 expression levels between IDC- DCIS nd pure IDC tumors nd the correltions with stndrd dignostic nd prognostic prmeters inside the IDC-DCIS group re unknown. Our im ws to determine if mir-21 hd the bility to distinguish these two invsive brest cncer groups. Levels of mir-21 expression were mesured by stem-loop quntittive Rel-Time PCR (RT-qPCR) method. Expression levels of estrogen receptor (ER), progesterone receptor (PR), humn epiderml growth fctor receptor 2 (Her-2) nd prolifertive index Ki-67 were evluted by immunohistochemistry. IDC-DCIS tumors hd significntly lower levels of mir-21 expression in grde 2 (P=0.003, Mnn-Whitney U test), ER positive (P=0.025, Mnn-Whitney U test) nd PR positive tumors (P=0.024, Mnn-Whitney U test) thn pure IDCs. mir-21 levels showed different pttern of expression in IDC-DCIS compred to IDC tumors, which is bsed on the difference in mir-21 expression between Her-2 negtive nd Her-2 positive IDC-DCIS tumors (P=0.030, Mnn-Whitney U test) nd high negtive correltion of mir-21 levels with PR levels (ρ=-0.886, P=0.006, Spermn correltion). According to our results, IDC-DCIS brest crcinoms ct in different mnner in pure IDC tumors with regrd to the reltions between mir-21 expression levels nd the stndrd dignostic nd prognostic prmeters, such s Her-2 sttus, ER nd PR sttus nd protein levels. Key words: Invsive ductl crcinom with non-invsive component (IDC-DCIS); mir-21 expression levels; the difference between IDC-DCIS nd IDC Received: Mrch 27, 2015; Revised: My 27, 2015; Accepted: June 02, 2015 INTRODUCTION Ductl crcinom in situ (DCIS) is brest cncer disese chrcterized by the cells tht proliferte inside the bsement membrne comprised of myoepithelil cells in the brest ducts (Virnig et l., 2010). In some cses, they ccompny invsive forms of the disese. About 25.4% cses of invsive brest crcinoms re ssocited with non-invsive component (Solimn et l., 2012), nd 30-60% ptients with IDC contin DCIS form (Dieterich et l., 2014). Solitry DCIS forms re very difficult to detect in Serbi becuse of the insufficient sensitivity of mmmogrphy to DCIS histology (Brem et l., 2007). Only one in 10-20 cses of dignosed brest cncer contins DCIS form (Verkooijen et l., 2003; Wong et l., 2010). IDC-DCIS re clssified ccording to the current Americn Joint Committee on Cncer stging system in the group of invsive brest crcinoms, bsed on the presence of invsive form (Singletry, 2002). Some 1285

1286 Petrović et l. studies hve demonstrted tht DCIS ssocited with invsive forms shre similr genomic profiles to pure invsive forms (Ikovlev et l., 2008). Although ptients with IDC-DCIS were clssified together with pure IDC ccording to its invsiveness (Chgpr et l., 2009), most reserchers nlyze IDC-DCIS nd pure IDC smples together. However, IDC-DCIS tumors on the moleculr level differ considerbly from pure IDC tumors, s Cstro et l. (2008) hve pointed out. Severl reserchers expect tumors contining both-idc nd DCIS components to develop less ggressive phenotype (Wong et l., 2010). Others suggest tht the presence of n in situ prt does not influence ptient prognosis t ll (Cstro et l., 2008). However, other reserchers developed n opposite hypothesis. Becuse of the diffuse nture of DCIS, fter the brest-conserving surgeries, IDC-DCIS ptients might hve greter chnce for locl recurrence thn pure IDC ptients, nd lso might hve worse prognosis (Chgpr et l., 2009). As the invsiveness of tumor is preliminry process to metstsis, it is now in the focus of mny studies. MicroRNA-21 hs been shown to be very powerful fctor in brest cncer invsion (Yn et l., 2008; Hung et l., 2009; Song et l., 2010; Tng et l., 2012; Petrović et l., 2014; Petrović et l., 2014b), but the expressionl levels of mir-21 nd its potentil correltions with the clinicl nd pthologicl fetures in IDC-DCIS tumors hve not been chrcterized yet. mir-21 is 22-nucleotide-long epigenetic fctor tht silences gene expression by binding to the mrnas of its trget genes (Qi et l., 2009). MicroRNA-21 is n oncogenic regultory element tht blocks or delys trnscription of tumor suppressor genes (Kim et l., 2009; Pn et l., 2010). This smll silencer of trnsltion is engged with six out of ten key moments in brest cncer development nd progression: growth suppressor evsion, prolifertive signling mintennce, resistnce to cell deth, tumor-promoting inflmmtion, induction of ngiogenesis, nd induction of invsion nd metstsis (Hnhn nd Weinberg, 2011), by trpping five trget-gene-mrnas: TIMP3, PDCD4, PTEN, TPM1 nd RECK (Yng et l., 2009; Buscgli, 2011). The IDC-DCIS form of brest cncer disese might be trnsitionl stte between in situ nd invsive brest crcinoms (Pinder nd Ellis, 2003). An IDC-DCIS tumor might occur de novo (Frbegoli et l., 2002; Ptl et l., 2002), or it cn rise s result of genetic nd epigenetic chnges in norml nd/or DCIS tissue (Velds et l., 2006; Wong et l., 2010). The tumor could lso be the result of chnges in the signling pthwys in neighboring cells of the tumor or norml tissue tht will be trnsformed to mlignnt (by prcrine regultion) (Polyk nd Hu, 2005; Dniel et l., 2011). The ssocition of mir-21 expression levels with stndrd dignostic nd prognostic prmeters in these entities is still unknown. It is lso unknown whether they ct in similr mnner to their potentil pure invsive reltives or the chnges occur s the consequences of ctions in different moleculr pthwys. In our previous study, we hve shown tht invsive with non-invsive ER positive tumors hve lower mir-21 expression thn pure invsive tumors (ILC, IDC nd mixed-ilc-idc tumors) nd higher thn non-invsive ER+ tumors (Petrović et l., 2014). We lso found tht invsive ssocited with non-invsive tumors hd lower expression of mir-21 in the PRpositive subgroup compred to both pure invsive nd even non-invsive tumors. Therefore, further reserch into nd chrcteriztion of these entities ws necessry. We performed n dditionl study to exmine their unusul behvior. Bsed on our previous reserch (Petrović et l., 2014b) nd ccording to mir-21 expression levels tht were distributed between non-invsive nd pure invsive brest cncers, we suggest tht IDC-DCIS might represent trnsitionl forms during brest cncer invsion nd progression. We hve continued investigting only IDC-DCIS cses to chrcterize their effect on mir-21 expression levels. In this study, we hve compred i) the mir-21 expression levels of 12 invsive ssocited with non-invsive brest crcinoms with 11 pure IDC tumors from the sme groups bsed on stndrd dignostic nd prognostic brest cncer prmeters; ii) the mir-21 expression levels within the group of IDC-DCIS tumors divided into

mir-21 expression in brest crcinom 1287 subgroups formed ccording to stndrd dignostic nd prognostic prmeters of brest tumors, nd iii) the mir-21 expression levels with DCIS contribution in IDC-DCIS brest crcinoms. Our bsic ssumption ws tht IDC-DCIS tumors differ from pure IDC brest crcinoms (lthough both groups re clssified s invsive brest crcinoms) in mir-21 expression levels in groups formed ccording to stndrd dignostic nd prognostic prmeters such s ge t dignosis, menstrul sttus, size of tumor, tumor grde, lymph node sttus, estrogen receptor (ER), progesterone receptor (PR), humn epiderml growth fctor receptor 2 (Her-2) nd prolifertion index Ki- 67. Our second ssumption (bsed on the experimentlly confirmed role of mir-21 in the process of brest cncer invsion) ws tht mir-21 expression levels negtively correlte with DCIS contribution. MATERIALS AND METHODS Study subjects During this reserch, we hve nlyzed 12 invsive brest cncer smples tht contined non-invsive components in different percent from ptients tht hd undergone surgery t the Institute for Oncology nd Rdiology of Serbi between June 2012 nd My 2013. We compred their mir-21 expression levels with 11 smples of pure invsive ductl crcinoms (selected ccording to similr dignostic nd prognostic prmeters to IDC-DCIS smples). Tumor smples were divided into two sections, nd chrcterized immeditely fter surgery. Prts of the tissues with t lest 75% of mlignnt cells were used for further moleculr nlysis nd prts were fixed with formlin nd embedded in prffin for routine pthologicl nd histologicl chrcteriztion, nlysis, nd rchiving. This study ws pproved by n Institutionl Review Bord pprovl ccording to the Ntionl Helth Regultion 5002/1-01, nd informed consent from ll the exmined ptients ws obtined. Histologicl type, histologicl nd nucler grde, lymph-node sttus were determined by two pthologists. ER, PR, Her-2 sttus nd Ki-67 were evluted by stndrd scoring system to determine dequte post-opertive therpy. IDC nd DCIS rtios in IDC-DCIS entities were presented in percentge determined by the two pthologists. Brest cncer tissue nd moleculr nlysis Immunohistochemistry For estrogen receptor (ER) nd progesterone receptor (PR) protein level evlution, rbbit nd mouse monoclonl ntibodies (RM-9101-S1, Thermo Fisher Scientific, Cheshire, UK, nd M3569, Dko Copenhgen, Denmrk, respectively) were used on 4-5-μm sections of formlin-fixed prffin-embedded blocks. The evlution of humn epiderml growth fctor receptor 2 (Her-2) sttus ws performed with ntibody rbbit-ntihumn A0485, (Dko, Copenhgen, Denmrk). Ki-67 prolifertion index ws chrcterized with RM9106-S1 (Thermo Fisher Scientific, Cheshire, UK). For ER nd PR sttus, Her-2 expression ws considered positive t 3+ by immunohistochemistry (IHC) nd t >2 by IHC nd positive by chromogenic in situ hybridiztion (CISH) if = 2 (2+) (Di Plm et l., 2006). Ki-67 levels were evluted s the percentge of positively stined cells. After deprffiniztion, rehydrtion nd tretment with 3% H 2 O 2 for 10 min, tissue slides were immersed in 10 mm of citrte buffer (ph 6) in microwve oven for 25 min. Smples were cooled nd wshed in TBS, ph 7.4. Sections were incubted with the ntibodies t 1:50 dilutions with Ab Diluent Buffer (Code No.S0809 Dko, Copenhgen, Denmrk). Then, smples were treted with the TP-125-HL (Thermo Fisher Scientific, Cheshire, UK) redy-to-use polymer system. For the visuliztion, we used diminobenzidine. Brown nucler stining in cells ws scored ccording stndrds from Leke (2000). RNA extrction nd purifiction All tissue smples were stored t -80 C immeditely fter surgery. Fresh-frozen tissues of brest cncer smples were homogenized in liquid nitrogen for fur-

1288 Petrović et l. ther RNA isoltion. Totl RNA ws extrcted from powdered tissues with TRI Regent (Ambion, Foster City). After 10-min incubtion in TRI Regent, 0.2 ml of chloroform ws dded to ech smple. The smples were centrifuged for 10 min t 14000 RPM (4 C) nd RNA smples were precipitted with isopropnol. Then, the smples were centrifuged (14000 RPM, 4 C) nd the pellet ws rinsed with 1 ml of 70% ice-cold ethnol. The pellets were dissolved in 100 μl DEPC-DW nd incubted t 65 C for 10 min. Quntifiction of RNA smples ws performed by Biospec-Nno (Shimdzu Corportion, Jpn). The qulity of RNA smples hs been lso confirmed by denturizing 1% grose gel electrophoresis. mir-21 reverse trnscription nd stem-loop qrt-pcr The expression of mture hs-mir-21 5p nd RNU6B s endogenous control ws mesured with TqMn Assys (ID: 000397 nd ID: 001093, respectively). For reverse trnscription we used in TqMn Micro RNA Reverse Trnscription Kit components nd the following therml conditions: 16 C for 30 min, 42 C for 30 min nd 85 C for 5 min. Following the steps from the TqMn Smll RNA Assys Protocol (Applied Biosystems, Foster City, CA), we performed reverse trnscription. In the second step, we quntified RTqPCR mplicons with Applied Biosystems (Foster City, CA) specific TqMn mir-21 nd RNU6B Assys with the following therml conditions: 95 C 10 min nd 95 C 15 s; 60 C 60 s for 40 cycles. MicroRNA-21 expression levels were presented in reltive units, nd the expression level vlues were normlized to RNU6B (smll nucler endogenous control), nd the smples were clibrted to the smple with the lowest reltive expression (set s referent-1x smple). Reltive quntity vlues were nlyzed with 7500 System SDS softwre. We used the reltive-quntity-δ Ct method, with the eqution: RQ smple =2 -(ΔCtsmple-ΔCtclibrtor) (where RQ smple represents reltive quntity of smple, while Ct = Ct mir- 21 -Ct RNU6B ). Sttisticl nlysis DCIS involvement ws presented by percents nd mir-21 reltive expression levels were chrcterized by their medins. We used the Mnn-Whitney U non-prmetric test to compre 2 independent groups of smples. For correltion nlysis, we used Spermn s. P vlues 0.050 were sttisticlly significnt, while those between 0.1 nd 0.05 were considered sttisticl trend. For the clcultions of P vlues, we used GrphPd Prism 5 softwre (GrphPd Softwre, Inc. CA). RESULTS mir-21 expression levels in IDC-DCIS nd IDC tumor groups We detected sttisticl trend in the difference in mir-21 expression levels between the group of IDC- DCIS nd pure IDC tumor smples (P=0.053, Mnn- Whitney U test). A sttisticlly significnt difference in mir-21 expression levels between IDC-DCIS nd pure IDC smples did not pper in ptients younger thn 60 yers, older thn 60 yers, postmenopusl ptients. Ten IDC-DCIS tumors differed with high significnce in mir-21 expression level from pure IDC grde 2 smples with P = 0.003 (Mnn-Whitney U test, Tble 1, Fig. 1A). In lymph node-positive nd lymph node-negtive tumors there ws no sttisticlly significnt difference in mir-21 expression level between IDC-DCIS nd IDC crcinoms ccording to the Mnn-Whitney U test. All IDC-DCIS ptients were postmenopusl with no difference in mir-21 expression compred with postmenopusl ptients with pure IDC tumors. In tumor smples with ER+ sttus, IDC-DCIS significntly differed from pure IDC tumor smples in mir-21 expression level with P=0.025 (Mnn-Whitney U test, Tble 1, Fig. 1B). In tumor smples with PR+ sttus, there ws lso sttisticlly significnt difference between IDC-DCIS nd pure IDC tumor smples in mirna-21 expression levels (P=0.024, Mnn-Whitney U test, Tble 1, Fig. 1C). In Her-2-negtive tumors, we detected

mir-21 expression in brest crcinom 1289 Tble 1. The difference in mir-21 reltive expression levels between size-mtched IDC-DCIS nd IDC tumor smples Reltive mir-21 expression levels Dignostic nd prognostic prmeters of brest tumors IDC-DCIS b N Pure IDC N c,d P vlue Age 60 50.817 (35.137-179.896) 4 239.382 (89.001-333.937) 8 P = 0.461 >60 50.895 (35.867-64.087) 6 90.459 (23.365-97.989) 3 P =0.548 Menstrul sttus Postmenopusl 99.775 (86.448-113.363) 8 88.802 (39.053-164.623) 4 P = 0.683 Grde 2 50.817 (35.867-64.087) 10 141.412 (97.989-270.900) 6 P = 0.003 Lymph node sttus Positive 61.777 (35.867-64.811) 6 95.479 (38.874-150.484) 6 P = 0.310 Negtive 50.817 (42.323-122.854 ) 6 141. 412 (58.097-231.405) 5 P = 0.537 ER sttus Positive 52.278 (35.867-64.268) 8 141.412 (87.121-231.405) 9 P = 0.025 PR sttus Positive 50.895 (34.779-64.449) 8 141.412 (87.121-231.405) 9 P = 0.024 Her-2 sttus Negtive 39.095 (33.691-45.088) 4 95.479 (56.956-145.948) 8 P = 1 Positive 64.087 (58.737-314.074 ) 6 239.382 (89.001-333.937) 3 P = 0.059 Ki-67 index (%) 20 64.449 (59.467-122.854) 7 150.484 (85.093-262.923) 5 P = 0.429 > 20 42.323 (37.481-44.397) 3 83.782 (36.807-141.412) 6 P = 0.381 Reltive mir-21 expression with 25th-75th percentile in prentheses. b N-number of smples. c P vlues equl or less thn 0.05 were considered significnt ccording to the Mnn-Whitney U test (between 2 groups). d P vlues less thn 0.1 were considered sttisticl trend. Fig. 1. The difference in mir-21 expression levels between IDC-DCIS nd pure IDC tumor smples in (A) grde 2 tumors; (B) ER positive subgroup; (C) PR positive subgroup. The vlues of mir-21 expression levels re shown in reltive units, normlized to RNU6B nd clibrted to the smple with lowest mir-21 expression. The plot shows interqurtile rnge (boxes) contoured with 25-75% of medin vlues. Middle line indictes the medin vlue nd the whiskers extended from the boxes represent the highest nd the lowest vlues, i.e. non-outlier rnges. Sttisticlly significnt differences were considered for P<0.05 vlues. sttisticl trend towrds incresed levels of mir-21 expression in pure IDC tumors compred with IDC- DCIS tumor smples (P=0.059, Mnn-Whitney U test, Tble 1). In Her-2-positive brest crcinom smples, sttisticlly significnt difference between IDC-DCIS nd pure IDC tumor smples ws not found. In ddition, there were no significnt differences in tumors with Ki-67 20% nd tumors with Ki-67 >20% between IDC-DCIS nd pure IDC tumors.

1290 Petrović et l. mir-21 expression levels in the IDC-DCIS tumor group In the group of ptients younger thn 60 yers, significntly different levels of mir-21 were not detected when compred with the group of ptients older thn 60 yers. Sttisticlly significntly higher mir-21 expression ws found in Her-2-positive compred to Her-2-negtive tumors with P=0.030 in the IDC- DCIS smples (Mnn-Whitney U test, Tble 2, Fig. 2A). Tumors with prolifertive index Ki-67 20% compred with Ki-67>20% tumors hd sttisticl trend towrds higher mir-21 expression levels in the IDC-DCIS smples (P=0.024, Mnn-Whitney U test Tble 2. The difference in mir-21 reltive expression levels between groups of IDC-DCIS tumor smples formed ccording to stndrd dignostic nd prognostic prmeters of brest tumors. Dignostic nd prognostic prmeters of tumors Age Reltive mir-21 expression in IDC-DCIS tumors 60 50.817 (35.137-179.896) 4 >60 50.895 (35.867-64.087) 6 d Size 2cm 42.323 (31.565-167.952) 5 > 2cm 59.467 (42.783-64.268) 7 Lymph node sttus Negtive 50.817 (42.323-122.854) 6 Positive 61.777 (35.867-64.811) 6 ER sttus Negtive 201.554 (56.547-346.561) 2 Positive 52.278 (35.867-64.811) 8 PR sttus Negtive 89.701 (50.870-234.707) 5 Positive 39.095 (33.691-59.467) 7 Her-2 sttus Negtive 39.095 (33.691-45.088) 6 Positive 64.087 (58.737-314.074 ) 4 Ki-67 index (%) 20 64.449 (59.467-122.854) 7 > 20 42.323 (37.481-44.397) 3 DCIS contribution (%) 30 64.449 (50.895-213.049) 4 > 30 50.817 (34.779-40.477) 8 b N c P vlue P = 1 P = 0.530 P = 0.937 P = 0.334 P = 0.283 P = 0.030 P = 0.024 P = 0.109 Reltive mir-21 expression with 25th-75th percentile in prentheses. b N-number of smples. c P Vlues equl or less thn 0.05 were considered significnt ccording to the Mnn-Whitney U test (between 2 groups). d Mximl tumor dimeter. Fig. 2. The difference in mir-21 expression inside the IDC-DCIS tumor group divided ccording to (A) Her-2 sttus nd (B) the vlues of Ki-67 prolifertion index. The vlues of mir-21 expression levels re shown in reltive units, normlized to RNU6B nd clibrted to the smple with lowest mir-21 expression. The plot shows interqurtile rnge (boxes) contoured with 25-75% of medin vlues. Middle line indictes the medin vlue nd the whiskers extended from the boxes represent the highest nd lowest vlues, i.e. non-outlier rnges. Sttisticlly significnt differences were considered for P<0.05 vlues. Tble 2, Fig. 2B). Significnt correltion between mir- 21 expression level nd the ge of ptients nd tumor size ws not found. MicroRNA-21 expression levels highly negtively correlted with PR sttus (ρ=-0.886, P=0.006, Spermn s correltion), but showed no correltion with ER, prolifertion index expression levels (Ki-67%) (Tble 3), nor with DCIS contribution in percentge (Tble 4). DCIS contribution in IDC-DCIS tumors Interestingly, there were no sttisticlly significnt differences in mir-21 expression between IDC-DCIS tumors with 30% of DCIS component compred with IDC-DCIS tht contined >30% DCIS. The distribution of DCIS in percentge positively correlted with tumor size, with high correltion coefficient vlue, ρ=0.718 (P=0.015, Spermn s correltion) (Tble 3).

mir-21 expression in brest crcinom 1291 Tble 3. Correltion of mir-21 expression levels with stndrd dignostic nd prognostic prmeters of tumors nd DCIS contribution. Vribles Number of smples Coefficient of correltion P vlue Age 10-0.353 0.387 Size 12 0 0.968 ER 10-0.206 0.578 PR 10-0.886 0.006 Ki-67 10 0.359 0.353 b DCIS % 12-0.418 0.297 ER, estrogen receptor; PR, progesterone receptor; Ki-67, prolifertion index. P-vlues less thn 0.05 were considered sttisticlly significnt. b Contribution of DCIS component. Tble 4. Correltion of DCIS contribution in percentge with stndrd dignostic nd prognostic prmeters of brest tumors. Vribles Number of smples Coefficient of correltion P vlue Age 10-0.089 0.785 Size 10 0.718 0.015 ER 10 0.344 0.283 PR 10-0.082 0.797 Ki-67 10-0.0289 0.924 ER, estrogen receptor; PR, progesterone receptor; Ki-67, prolifertion index. P-vlues less thn 0.05 were considered sttisticlly significnt. b Contribution of DCIS component. Tble 4. Correltion of DCIS contribution in percentge with stndrd dignostic nd prognostic prmeters of brest tumors. Vribles Number of smples Coefficient of correltion P vlue Age 10-0.089 0.785 Size 10 0.718 0.015 ER 10 0.344 0.283 PR 10-0.082 0.797 Ki-67 10-0.0289 0.924 ER, estrogen receptor; PR, progesterone receptor; Ki-67, prolifertion index. P-vlues less thn 0.05 were considered sttisticlly significnt. b Contribution of DCIS component. In our experiment, DCIS contribution in IDC-DCIS entities correlted neither with ptient ge, ER nd PR sttus, nor with the percentge of prolifertion index Ki-67 expression levels (Tble 4). DISCUSSION IDC-DCIS re clssified ccording to the Americn Joint Committee in Cncer stging system s pure invsive forms (Singletry, 2002). Some studies hve demonstrted tht DCIS ssocited with invsive forms shre similr genomic profiles with pure DCIS (Ikovlev et l., 2008), but Ctro et l. (2008) showed tht IDC-DCIS tumors significntly differ from IDC brest cncers t the moleculr level. The concept nd the ide of our pproch derived from our previous reserch where we noticed n interesting behvior of invsive brest crcinoms with non-invsive component in tht they significntly differed from pure invsive forms ccording to mir- 21 expression in the groups formed ccording to ER, PR, grde 2, nd K-67 20%. However, in the first study, we nlyzed ll types of pure invsive crcinoms together (invsive lobulr, ductl nd mixed). In order to highlight the significnce of these unique entities nd to emphsize the potentil influence of certin prognostic nd dignostic fctors, vi chnges in mir-21 expression levels, we excluded ll types of brest crcinom (BC) tht hd originted from other thn brest ducts. Additionlly, we included only IDCs with similr sizes to IDC-DCIS. In this study, we incresed the number of IDC-DCIS smples. The novelty of this work lies in the fct tht no reserch to dte, to the best of our knowledge, hs nlyzed the ssocition of mir-21 expression levels with stndrd dignostic nd prognostic prmeters inside the IDC- DCIS group. In ddition, no one hs yet compred DCIS contribution (s potentil fctor tht might be relted to BC invsion) with the fctor of BC invsiveness such s mir-21. We observed sttisticl trend towrds incresed levels of mir-21 expression in 11 pure IDC smples compred with 12 IDC-DCIS tumors. In grde 2-IDC

1292 Petrović et l. nd IDC-DCIS tumors, the sttisticlly significnt difference ppered to be very high (with low P vlue). When we compred high mir-21 IDC-DCIS with high mir-21 IDC (ccording to their medin rnges), we observed tht the IDC tumors hve significntly higher mir-21 levels, wheres smples with low mir-21 levels did not differentite between IDC- DCIS nd IDC smples. Additionlly, this might be specil trget group: high mir-21-idc-dcis or IDC-grde 2 group for future strtifiction nd clssifiction of brest crcinom ptients. MicroR-21 expression levels found in IDC-DCIS tumors did not correlte with ER sttus, which is unusul, becuse in the group of pure invsive tumors, levels of mir-21 expression correlted highly positively with the levels of ER expression, which ws lso shown by Petrović et l. (2014). In ddition, ER+ nd PR+ IDC-DCIS significntly differed from IDC ER+ nd PR+ tumors in mir-21 expression levels. Although it hs been shown tht mir-21 expression might be process dependnt on ER expression (Mttie et l., 2006; Wickrmsinghe et l., 2009; Petrović et l., 2014), in IDC-DCIS tumors, this might not be the cse. As we expected, difference in mir-21 expression levels in PR+ ptients ppered between the IDC-DCIS nd IDC tumor groups, which indictes tht higher mir-21 expression lso might not be inducted by incresed levels of progesterone receptor. Surprisingly, the mir-21 levels of IDC-DCIS tumors highly negtively correlted with PR sttus, while pure invsive BCs from our previous study (Petrović et l., 2014) highly positively correlted with PR levels. The fct tht mir-21 levels do not correlte with ge, while in pure invsive they correlte negtively with the ge of ptients (Petrović et l., 2014b), could be due to the bsence of ER/PR regultion of mir-21 expression in IDC-DCIS tumor specimens becuse of the differences in hormonl sttus relted to ptient ge (M et l., 2006). In IDC-DCIS tumors, Her-2 positive tumors hd significntly higher levels of mir- 21 expression, while pure invsive (IDC nd ILC) tumors cted independently from Her-2 sttus in the previous nlysis (Petrović et l., 2014). Hung et l. (2009) described Her-2-dependent mir-21 expression. We hve lso found tht IDC-DCIS tumors do not hve ER- nd/or PR-dependnt mir-21 expression, so these cses of multi-component tumors might hve Her-2-dependnt expression of mir-21, which supports our finding tht there ws significnce in the difference of mir-21 expression levels between Her- 2-negtive nd Her-2-positive tumors. We detected significnt difference in mir-21 expression levels between IDC-DCIS nd IDC Her-2-positive tumors. This mens tht the Her-2 receptor is nother fctor tht seprtes IDC-DCIS nd IDC tumors with regrd to mir-21 expression (Hung et l., 2009). Ki-67 is mrker of tumor ctivity. Higher Ki-67 levels might indicte potentil progression from non-invsive towrds ggressively invsive phenotype (Gerdes et l., 1991). Elevted Ki-67 levels in DCIS tumors might nticipte recurrence of the in situ crcinom fter brest-conserving surgery or progression to IDC. Wong et l. (2010) showed tht the frequency of IDC- DCIS ws ssocited with Ki-67 levels in low Ki-67 group. We did not find sttisticlly significnt differences between mir-21 levels in IDC-DCIS nd IDC groups when divided into tumors with Ki-67 20% nd Ki-67>20%. In the IDC-DCIS tumor group, we observed tht tumors with Ki-67 20 hd significntly higher mir-21 expression levels thn Ki-67>20 tumors, similr to pure invsive tumors from our previous study, (Petrović et l., 2014). Our findings could be explined by the dul form of the tumor, i.e. the influences of both-dcis nd IDC components tht re individul. Wong et l. (2010) implied in their reserch similr findings. We ssumed tht the presence of DCIS might not be silent, nd might hve some impct bsed on the chnges in genetic nd epigenetic levels tht might hve some repercussion on the behvior of the entire tumor (Chgpr et l., 2009). However, our findings showed tht there were no reltions between mir-21 expression levels nd DCIS contribution. Furthermore, we compred IDC-DCIS tumors with less thn or equl to 30% of DCIS with tumors contining more thn 30% of DCIS, nd there ws lck of significnt difference. DCIS % highly positively correlted with tumor size, but correltions with ge, ER, PR or Ki-67 were not found. Also,

mir-21 expression in brest crcinom 1293 mir-21 expression does not depend on the histologicl type of tumor (whether we compre IDC-DCIS with IDC, with ILC, or ILC nd IDC together) ccording to the results of previous nd present studies (Petrović et l., 2014; Petrović et l., 2014b). The question whether these re trnsitionl forms between in situ nd invsive tumors is impossible to nswer, becuse ech person is unique nd brest cncer is very heterogeneous disese, influenced by mny genetic, epigenetic nd microenvironmentl fctors (Allinen et l., 2004; Polyk nd Hu, 2005; Polyk, 2007). In our previous reserch (Petrović et l., 2014) we implied tht it ws necessry to dd new non-stndrd dignostic nd prognostic mrkers nd tht it ws lso necessry to form dditionl systems of clssifiction nd therpeutic pproch. New therpy pproches should be used especilly in ER+/PR+ nd ER+/ PR- groups of ptients with higher mir-21 expression levels, in order to prevent or hlt further invsion nd metstses in ptients tht do not respond or re resistnt to tmoxifen. It hs lredy been shown tht mir-21 expression ws relted to ER expression (Wickrmsinghe et l., 2009; Yn et l., 2011). Also, mir-21 expression levels in ER+/PR+, ER+PR- or ER-/PR- subgroups of ptients with invsive BCs ssocited with non-invsive should be considered to ct differently. These findings confirm our ssumption tht invsive cncers with non-invsive brest cncer forms ct in different mnner when compred to pure invsive tumors ccording to mir-21 expression levels. In ddition, bsed on our findings relted to the different behvior of mir-21/er/pr/her-2 fctors, high- mir-21/er+/pr+/pr-/grde 2 nd/or Her-2+ IDC-DCIS tumors might hve therpeuticlly chllenging phenotype for future nti-mir therpies. MicroRNA-21 expression in IDC-DCIS tumors tht were investigted might hve been Her-2-dependnt; this could hve been responsible for the difference in mir-21 expression between Her-2- nd Her- 2+ subgroups (tht ws not detected in pure invsive tumors) (Hung et l., 2009; Petrović et l., 2014; Petrović et l., 2014b). Thus, Her-2+ groups with higher mir-21 expression levels in ptients tht do not respond to conventionl therpies with herceptin should be considered for future nti-mir-21 therpy (in contrst to pure invsive brest crcinoms, ccording to our study). In summry, in the previous reserch we showed tht mir-21 expression in groups formed ccording to ER, PR, grde 2 nd K-67 20% significntly differed from those in pure invsive forms, while in our novel reserch (with fewer fctors included nd with slightly lrger IDC-DCIS group), these two groups differed with higher significnce in our present reserch, nd differed in Her-2-positive tumors, unlike in our previous reserch (Petrović et l., 2014b). Also in this study, the difference in K-67 20% group did pper, unlike previous results (Petrović et l., 2014b). CONCLUSIONS This study shows the complexity nd heterogeneity of brest cncer nd the need for dditionl systems of clssifiction s well s the need to identify new fctors/biomrkers. In order to improve ccess to tretment nd therpy, multi-component tumors should be considered s specil entities. It is necessry to seek new wys of tretment nd to move towrds personlized nd wy from generlized pproch to ptients with brest crcinom. Acknowledgments: This work ws supported by the Ministry of Eduction nd Science, Republic of Serbi, Grnt ON173049. Conflict of interest disclosure: All uthors declre tht they hve no conflict of interest. REFERENCES Allinen, M., Beroukhim, R., Ci L., Brennn, C., Lhti-Domenici, J., Hung, H., Porter, D., Hu, M., Chin, L., Richrdson, A., Schnitt, S., Sellers, W.R. nd K. Polyk (2004). Moleculr chrcteriztion of the tumor microenvironment in brest cncer. Cncer Cell. 6, 17-32. Brem, R. F., Fishmn M. nd J.A. Rpelye (2007). Detection of ductl crcinom in situ with mmmogrphy, brest spe-

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