It is suggested that tumour stromal myofibroblasts exert an unfavourable effect on the biology of breast cancer. We are aware of only a single study which examined relationships between manifestation of myofibroblasts in the stroma of breast cancer and clinicopathological data of the patients. The present study was aimed at estimation of the effect exerted by myofibroblasts present in the tumour stroma on principal pathological parameters and on expression of Ki67, P53 and HER-2 proteins in the group of the most frequent breast cancers, the ductal cancers. In paraffin sections of 60 ductal breast cancers (20 cases in G1, 20 in G2 and 20 in G3), immunohistochemical reactions were performed to detect expression of smooth muscle actin (SMA) in order to visualize myofibroblasts, Ki67, P53 and HER-2. The studies demonstrated that the most numerous myofibroblasts were present in G3 cases and they were the least frequent in G1 cases (P = 0.02). Positive correlations were observed between the presence of myofibroblasts in tumour stroma and expression of Ki67 and HER-2 in breast cancer cells in the entire group (P < 0.001 and P = 0.001, respectively), in G2 cases (P = 0.003 and P = 0.03) and in G3 cases (P = 0.01 and P = 0.03). Considering that the higher grade, Ki67 and HER-2 are thought to represent unfavourable prognostic factors, the elevated content of myofibroblasts in tumour stroma is probably typical for cases with worse prognosis

Gansukh, Tserenchunt

GyĂśrffy, Balazs

Maciejczyk, Adam

Pudełko, Marek

Suchocki, Sławomir

Surowiak, Paweł

Wojnar, Andrzej

Zabel, Maciej

English

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 44, No. 2, 2006pp. 111-116Stromal myofibroblasts in breast cancer: relationsbetween their occurrence, tumor grade and expression of some tumour markersPaweł Surowiak1,2,3, Sławomir Suchocki4, Balazs Györffy2,5, Tserenchunt Gansukh2,6,Andrzej Wojnar3, Adam Maciejczyk3, Marek Pudełko3 and Maciej Zabel1,71Department of Histology and Embryology, University School of Medicine, Wrocław, Poland; 2Institute of Pathology, Charité Campus Mitte, Berlin, Germany; 3Lower Silesian Centre of Oncology, Wrocław, Poland; 4Department of Obsterics and Gynaecology, Voivodship Hospital, Wałbrzych, Poland; 5Joint Laboratory of the Hungarian Academy of Sciences and the Semmelweis University,  2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary; 6Health Sciences University of Mongolia, Ulaanbaatar, Mongolia; 7Department of Histology and Embryology, University School of Medicine, Poznan, PolandAbstract: It is suggested that tumour stromal myofibroblasts exert an unfavourable effect on the biology of breast cancer. Weare aware of only a single study which examined relationships between manifestation of myofibroblasts in the stroma of breastcancer and clinicopathological data of the patients. The present study was aimed at estimation of the effect exerted bymyofibroblasts present in the tumour stroma on principal pathological parameters and on expression of Ki67 , P53 and HER-2proteins in the group of the most frequent breast cancers, the ductal cancers. In paraffin sections of 60 ductal breast cancers(20 cases in G1, 20 in G2 and 20 in G3), immunohistochemical reactions were performed to detect expression of smooth muscleactin (SMA) in order to visualize myofibroblasts, Ki67, P53 and HER-2. The studies demonstrated that the most numerousmyofibroblasts were present in G3 cases and they were the least frequent in G1 cases (P=0.02). Positive correlations wereobserved between the presence of myofibroblasts in tumour stroma and expression of Ki67 and HER-2 in breast cancer cellsin the entire group (P <0.001 and P=0.001, respectively), in G2 cases (P=0.003 and P=0.03) and in G3 cases (P=0.01 andP=0.03). Considering that the higher grade, Ki67 and HER-2 are thought to represent unfavourable prognostic factors, the elevatedcontent of myofibroblasts in tumour stroma is probably typical for cases with worse prognosis. (www.cm-uj.krakow.pl/FHC)Key words: Myofibroblasts - Breast cancer - Grade - Ki67 - P53 - HER-2IntroductionBreast cancer is the most common malignant tumour offemales in the western world, being responsible forabout 32% of the estimated new female cancer cases.The incidence of breast cancer remains high, and theclinical courses are highly variable. For years, a growingincidence of the disease has been documented and therisk of dying of breast cancer continues to increase inconsecutive cohorts of generations [5]. Therefore, innumerous centres intense efforts develop to detect newprognostic factors which would permit to intensify thetherapy in high risk cases. Moreover, such prognosticfactors might provide an interesting topic for studies onnew therapeutic approaches. Conventional prognosticand predictive markers for breast cancer are stage of theadvancement (nodal status, tumour size and presence ofthe metastases), tumour grade and tumour size [4]. Ex-pression of receptors for female sex hormones [10] andHER-2 is also of importance [4]. In addition, expressionof the proliferation-associated antigen, Ki67 and of thesuppressor protein, P53 are frequently estimated in orderto establish the dynamics of tumour growth [12]. Signific-ance of several other proteins and of their expression inbreast cancer cells continues to stimulate studies world-wide. Until now, however, only expression of receptors forCorrespondence: P. Surowiak, Dept. Histology and Embryology,University School of Medicine, Chałubinskiego 6a, 50-356 Wrocław, Poland ; e-mail: pawel.surowiak@interia.plfemale sex hormones and of HER-2 have found appli-cation in a routine diagnosis of breast cancer [4]. In recent years, numerous publications have de-scribed effects of tumour-associated tissues on thedynamics of neoplastic disease. Invasion of tumourcells is slowed down by myoepithelial cells and basalmembranes [9]. The immune system has been foundto protect the body from neoplastic cells. On the otherhand, tumour cells produce, i.a. metalloproteinasesand growth factors which may facilitate invasion oftumour cells. Tumour-associated vessels provide path-way for spread of tumour cells [6]. The interactionsbetween tumour cells and stromal myofibroblasts re-main, however, poorly recognized. Myofibroblasts pro-duce several factors which may stimulate proliferationof cancer cells and facilitate their infiltration. They havebeen found to secrete, i.a., insulin-like growth factor-2(IGF-2) and hepatocyte growth factor (HGF) [2]. Recog-nition of relationships between myofibroblasts and breastcancer cells may facilitate definition of new prognosticfactors and provide targets for novel therapeutic ap-proaches.The present study was aimed at examination of rela-tionships between the presence of myofibroblasts intumour stroma and principal pathological parameters aswell as expression of Ki67, P53 and HER-2 proteins ina group of most frequent breast cancers, the ductalcancers.Materials and methodsPatients. Immunocytochemical analysis was performed retrospec-tively on tissue samples collected for routine diagnostic purposes.Based on histology (invasive ductal breast cancer) and grade (equalgroups for each grade), 60 patients with primary invasive breastcancer diagnosed in the years 1999 to 2000 in the Lower SilesianCentre of Oncology (Wroclaw, Poland) were included in the study.The mean age of the patients was 55.62 years ± 9.86 SD (age range:43 to 72 years). Every grade (G1, G2 and G3) was represented by 20cases. In 34 patients stage 2a, in 22 patients stage 2b and in 4 patientsstage 3 of the tumour was diagnosed. Fragments sampled from thestudied tumours were fixed in 10% buffered formalin and embeddedin paraffin. In every case, hematoxylin and eosin stained sectionswere examined by two pathologists. The stage of tumours wasassessed according to TNM classification system [8]. Tumour gradewas estimated according to Bloom-Richardson, according to themodification of Elston and Ellis [3] (Table 1).Immunohistochemistry. Formalin-fixed, paraffin-embedded sec-tions (4 µm) were mounted on Superfrost slides (Menzel Gläser,Germany), dewaxed with xylene, and gradually rehydrated. Activityof endogenous peroxidase was blocked by 30 min incubation in 1%H2O2. The studied sections were boiled 15 minutes in Target Retrie-val Solution (DakoCytomation, Poland) , in a microwave oven at 250W. Then, immunohistochemical reactions were performed usingmonoclonal antibodies detecting smooth muscle actin (SMA, clone1A4, DakoCytomation, Poland) diluted 1:200, Ki67 (clone MIB-1,DakoCytomation, Poland) diluted 1:100, P53 (clone DO-7, Dako-Cytomation, Poland) diluted 1:100 and polyclonal anti-HER-2 anti-bodies (DakoCytomation, Poland) diluted 1:350. The antibodieswere diluted in the Antibody Diluent, Background Reducing (Dako-Cytomation, Denmark). The sections were incubated with antibodiesfor 1 h at room temperature. Subsequent incubations included bioti-nylated secondary antibodies (15 min, room temperature) and strep-tavidin-biotinylated peroxidase complex (15 min, room temperature)(LSAB+, HRP, DakoCytomation, Denmark). NovaRed (Vector La-boratories, UK) was used as chromogen (10 min, at room tempera-ture). All sections were counterstained with Meyers hematoxylin.In every case, control reactions were included, in which specificantibody was substituted by the Primary Mouse Negative Control(DakoCytomation, Denmark).Scoring of immunostaining results. Myofibroblasts visualized bySMA expression were assessed semiquantitatively with the use of asimplified scale based on the percentage of myofibroblasts in thepopulation of tumour stromal cells (0 - no myofibroblasts, 1 - <10%of myofibroblasts in tumour stroma, 2 - 10-30% of myofibroblastsin tumour stroma, 3 - >30% of myofibroblasts in tumour stroma).Expression of P53 and Ki67 was quantitated by calculating thepercentage values of immunopositive cells. For evaluation of HER-2reactivity, the DakoCytomation scoring system was used (0 = nega-tive; + = partial membranous; ++ = complete membranous, weak;+++ = complete membranous, strong). The intensity of immunohis-tochemical reactions was assessed independently by two patholog-ists; in doubtful cases a re-evaluation was performed using adouble-headed microscope. In each case we analyzed 6 microscopicfields at × 200 magnification.Statistical analysis. Statistical analysis of the obtained results wasconducted using Statistica 98 PL software (Statsoft, Poland). At thefirst stage of the calculations, the presence of myofibroblasts wascompared in individual groups (G1, G2, G3) using Kruskal-Wallisrank ANOVA test. Subsequently, relationships were examined (em-ploying the same test) between the presence of myofibroblasts andexpression of the studied variables in all groups together (G1 to G3)and in each group (G1, G2 and G3). ResultsImmunostainingDetection of SMA yielded reaction of a cytoplasmiclocalization and of variable intensity in individual cases.SMA was present in myofibroblasts (Fig. 1), myoepithe- Table 1. Characteristics of patients and tumoursCharacteristics No. (%)Patients 60 (100)Age (mean 55.62years ± 9.86 SD)≤50  5 (8.3)50-60 32 (53.3)>60 23 (38.3)Tumour grade1 20 (33.3)2 20 (33.3)3 20 (33.3)Tumour stage2a 34 (56.7)2b 22 (36.7)3  4 (6.6)HistologyInvasive ductalbreast cancer60 (100)  112  P. Surowiak  et al.lial cells and in muscles of blood vessels. According tothe applied scale, the mean manifestation of myofibro-blasts amounted to 2.01±0.69.Application of antibodies against P53 or Ki67yielded in tumour cells colour reaction of nuclear local-ization and of a variable intensity in individual cases.The mean percentage of P53-positive and Ki67-positivebreast cancer cells was 40.71±26.69 and 28.56±17.7,respectively.In the case of HER-2, membrane reaction was ob-tained of various intensity in individual cases, localizedin the tumour cells. The mean reaction intensity inDakoCytomation 0-3 score was 1.84±0.74.Relationships between the presence of myofibroblasts and histopathological parametersThe analysis performed using the Kruskal-Wallis rankANOVA test demonstrated no relationships between thepresence of myofibroblasts in tumour stroma and stageor age of the studied patients (P=0.67 and P=0.54,respectively). Nevertheless, a significant positive corre-lation was disclosed between the presence of myofibro-blasts in tumour stroma and grade of the studied tumors(P=0.02, Fig. 2). Cases of higher grade manifestedaugmented content of myofibroblasts in tumour stroma.Relationships between the presence of myofibroblasts and expression of P53, Ki67 and HER-2Using also the Kruskal-Wallis rank ANOVA test, rela-tionships were examined between the presence ofmyofibroblasts in the tumours and expression of P53,Ki67 and HER-2 in individual grades (G1, G2 and G3)or in the entire group (G1-3). The test demonstrated nosignificant relationships in the G1 group (P>0.05, Table 2).In none of the examined groups could a relationship bedisclosed between the presence of myofibroblasts in theFig. 1. Immunohistochemical localization of smooth muscle actin inbreast cancer tumors. A. Reaction in individual myofibroblasts(<10%, score: 1) in tumour stroma (gray arrow) and in blood vessel(black arrow). × 200. B. Reaction in numerous myofibroblasts(>30%, score: 3) in tumour stroma (gray arrow). × 200. C. Reactionin numerous myofibroblasts (>30%, score: 3) in tumour stroma (grayarrow). × 400.Fig. 2. The presence of myofibroblasts in tumour stroma related totumour grade (Kruskal-Wallis rank ANOVA test). Myofibroblastsare the most numerous in stroma of G3 tumours.Myofibroblasts and breast cancer  113tumour stroma and expression of P53 in cancer cells(P>0.05, Table 2). However, in groups G2 (Fig. 3), G3(Fig. 4) and in the entire studied material (G1-3, Fig. 5)a significant positive correlation was noted between thepresence of myofibroblasts in tumour stroma and ex-pressions of Ki67 and HER-2 in cancer cells (P<0.05,Table 2). In cases with the highest content of myofibro-blasts in tumour stroma, the most pronounced express-ion of  Ki67 and HER-2 was also observed in cancercells.DiscussionThe present study demonstrates the significance ofmyofibroblast presence in tumour stroma, as detected bysmooth muscle actin (SMA) expression [1]. We havedocumented that myofibroblasts are present in tumourstroma in a proportion of breast cancers. This result hasconfirmed results of other authors. Yazhou et al. [11]investigated the relation between the presence of myofi-broblasts in tumour stroma and clinico-pathologicalvariables in the breast cancer patients. In the presentstudy we have shown that cases of higher grade demon-strate higher content of myofibroblasts in tumour stro-ma. Similarly to Yazhou et al. [11], we have noted norelationship between manifestation of myofibroblastsand age of the studied patients. Also, in line with the dataof the cited authors, the content of myofibroblasts intumour stroma has not been related to stage of thestudied tumours. We have also examined potential relationships be-tween manifestation of myofibroblasts in tumour stromaand expression of Ki67 , P53 and HER-2 in breast cancercells. Until now, only the relationship between myofi-broblast content and expression of Ki67 in breast cancercells has been investigated [11] and no significant rela-tionship has been detected. In the present study, how-ever, cases with higher content of myofibroblasts intumour stroma have been found to contain higher pro-portions of cancer cells with Ki67 expression. Sincemyofibroblasts, both in  tumours and in healing pro-cesses, produce numerous growth factors [2], we sug-gest that myofibroblast-released growth factors mightstimulate proliferation of breast cancer cells. In this study, no relationship has been documentedbetween the presence of myofibroblasts in tumour stro-ma and expression of P53 in breast cancer cells. How-ever, cases with higher content of myofibroblasts haveFig. 4. In G3group, cases with the highest content of myofibroblastsexhibit: A. Higher content of cancer cells with Ki67 expression; B.Higher expression of HER-2 in cancer cells (Kruskal-Wallis rankANOVA test).Fig. 3. In G2 group, cases with the highest content of myofibroblastsexhibit: A. Higher content of cancer cells with Ki67 expression; B.Higher expression of HER-2 w in cancer cells (Kruskal-Wallis rankANOVA test). 114  P. Surowiak  et al.been found to exhibit more intense expression of HER-2in breast cancer cells. Myofibroblasts may secrete in-sulin-like growth factor-2 (IGF-2) and hepatocytegrowth factor (HGF) [7]. They represent ligands forreceptors of the Epidermal Growth Factor Receptorgroup, which includes, i.a., HER-2. The ligands, mostprobably in a paracrine way, stimulate expression ofHER-2 in breast cancer cells. Since HER-2 representsone of the best recognised negative prognostic factors[4] we suggest that the presence of myofibroblasts intumour stroma may be linked to unfavourable prognosisin breast cancer patients. In summary, the present investigations have demon-strated that breast cancer cases with higher content ofmyofibroblasts in tumour stroma are characterised byhigher grade of the tumour, more intense proliferationof neoplastic cells and more intense expression of HER-2 in cancer cells. To our knowledge, this paper describesfor the first time a positive correlation between thepresence of myofibroblasts in tumour stroma and intens-ity of Ki67 and HER-2 expression. Taking into accountthat higher grade, Ki67 and HER-2 are regarded torepresent negative prognostic indicators, the intensifiedcontent of myofibroblasts in tumour stroma may betypical for cases with a worse prognosis. The signific-ance of myofibroblast presence in the stroma of breastcancers should be investigated in detail, in order to detectnew prognostic indicators and novel targets for thera-peutic approaches.References[ 1] Chaponnier C, Gabbiani G (2004) Pathological situations char-acterized by altered actin isoform expression. J Pathol 204:386-395[ 2] De Wever O, Mareei M (2003) Role of tissue stroma in cancercell invasion. J Pathol 200: 429-447[ 3] Elston CW, Ellis IO (1991) Pathological prognostic factors inbreast cancer. The value of histological grade in breast cancer:experience from a large study with long-term follow-up. Histo-pathology 5: 403-410[ 4] Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC,Clark GM, Ruby SG, OMalley F, Simpson JF, Connoly JL,Hayes DF, Edge SB, Lichter A, Schnitt SJ (2000) Prognosticfactors in breast cancer. College of American Pathologists Con-sensus Statement 1999. Arch Pathol Lab Med 124: 966-978[ 5] Jemal A, Thomas A, Murray T, Thun M (2002) Cancer statistics.CA Cancer J Clin 52: 23-47[ 6] Mori I, Yang Q, Kakudo K (2002) Predictive and prognosticmarkers for invasive breast cancer. Pathol Inst 52: 186-194[ 7] Offersen BV, Nielsen BS, Hoyer-Hansen G, Rank F, Hamilton-Dutoit S, Overgaard J, Andreasen PA (2003) The myofibroblastis the predominant plasminogen activator inhibitor-1-express-ing cell type in human breast carcinomas. Am J Pathol 163:1887-1899[ 8] Sobin LH, Wittekind C (2002) TNM classification of malignanttumours. 5th edition. Wiley-Liss INC., New York[ 9] Surowiak P, Dziegiel P, Matkowski R, Kornafel J, Wojnar A,Zabel M (2002) Immunocytochemical evaluation of metallo-thionein (MT) expression in myoepithelial cells of ductal mam-mary carcinoma and its relation to survival time: analysis ofTable 2. Correlations between the presence of myofibroblasts intumour stroma (SMA) and expression intensity of the studied anti-gens in breast cancer cells in the entire group (G1-3) and in the G1,G2 and G3 groups (Kruskal-Wallis rank ANOVA test).Studied pair of variablesGrade P valueSMA and P53G1-3 0.5399G1 0.2625G2 0.2521G3 0.3957SMA and Ki67G1-3 <0.001G1 0.1111G2 0.0031G3 0.0114SMA and HER-2G1-3 0.0011G1 0.2083G2 0.0299G3 0.0349        Fig. 5. In the entire material (G1-3), cases with higher content ofmyofibroblasts exhibit: A. Higher proportion of cancer cells withexpression of Ki67; B. Higher expression of HER-2 in cancer cells(Kruskal-Wallis rank ANOVA test).Myofibroblasts and breast cancer  1157-year course of the disease. Folia Histochem Cytobiol 40:199-200[10] Surowiak P, Dziegiel P, Zabel M, Matkowski R, Kornafel J(2001) Prognostic value of immunocytochemical estimation ofestrogen receptor (ER) and of pS2 estrogen-dependent proteinin cells of mammary ductal carcinoma. Analysis of five-yearcourse of the disease. Folia Histochem Cytobiol 39: 143-144[11] Yazhou C, Wenlv S, Weidong Z, Licun W (2004) Clinicopa-thological significance of stromal myofibroblasts in invasiveductal carcinoma of the breast. Tumor Biol 25: 290-296[12] Zasławski R, Surowiak P, Paluchowski P, Dziegiel P, Ma-ciejczyk A, Pudełko M, Wojnar A, Zabel M (2005) Differencesin oestrogen and progesterone receptors, HER-2, p53 express-ion and proliferation in ductal breast cancers in relation tohistopathological grade. Folia Morphol 64: 9-15      Received: October 14, 2005Accepted after revision: December 14, 2005 116  P. Surowiak  et al.

Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers.

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Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers.

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