Fibroadenoma is the most prevalent benign breast tumor. It consists of epithelial and stromal components. In general, breast tumors are highly hormonally dependent and growth hormone by its physiology may have a possible oncogenic potential. Therefore, the aim of this study was to determine the expression of growth hormone and growth hormone receptor in epithelial and stromal components of fibroadenomas. Study group included 30 randomly chosen fibroadenomas from female patients aged between 18 and 69 years. The expression of growth hormone and growth hormone receptor was defined in both histologic components of fibroadenomas. Growth hormone was expressed in 96.7% of both epithelial and stromal components of fibroadenomas, with stronger expression in the stromal component. The same percentage of positive reaction (96.7%) was obtained in the epithelial component of fibroadenomas for growth hormone receptor expression. Only 6.7% of stromal components tested for growth hormone receptor were positive. The high expression of growth hormone and growth hormone receptor in fibroadenoma tissue indicates their possible role in the pathogenesis of this tumor. Follow up of patients with high expression of growth hormone and growth hormone receptor may be suggested.Fibroadenom je najčešća dobroćudna novotvorina dojke, a sastoji se od epitelne i stromalne komponente. Uzimajući u obzir hormonsku osjetljivost tumora dojke, te potencijalni onkogeni potencijal hormona rasta, cilj našega rada bio je utvrditi ekspresiju hormona rasta i receptora hormona rasta u epitelnoj i stromalnoj komponenti fibroadenoma. U studiju je bilo uključeno 30 nasumice odabranih fibroadenoma nastalih u žena u dobi od 18 do 69 godina. Ekspresija hormona rasta i receptora hormona rasta određivana je u obje histološke komponente fibroadenoma. Ekspresija hormona rasta nađena je u 96,7% epitelne i stromalne komponente, uz jaču izraženost ekspresije u stromalnoj komponenti. Ekspresija receptora hormona rasta u epitelnoj je komponenti također iznosila 96,7%, a u stromalnoj samo 6,7%. Visoka ekspresija hormona rasta i receptora hormona rasta u fibroadenomima označava potencijalnu ulogu ovoga hormona u patogenezi ispitivane bolesti, te bi se u bolesnica ove skupine mogle preporučiti češće kliničke kontrole

Božo Krušlin

Dino Kasumović

Emil Štajduhar

Tanja Leniček

Tihana Džombeta

Zoran Jukić

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Acta Clin Croat,  Vol. 52,  No. 2,  2013 235Acta Clin Croat 2013; 52:235-239 Professional PaperExPrEssion of growth hormonE And growth hormonE rECEPtor in fibroAdEnomAs of thE brEAsttanja Leniček1, dino Kasumović2, Emil Štajduhar3, tihana džombeta2, Zoran Jukić4 and božo Krušlin1,21Ljudevit Jurak department of Pathology, sestre milosrdnice University hospital Center; 2department of Pathology, school of medicine, University of Zagreb; 3department of radiology, sestre milosrdnice University hospital Center, Zagreb; 4department of surgery, nova gradiška general hospital, nova gradiška, CroatiasUmmArY – fibroadenoma is the most prevalent benign breast tumor. it consists of epithelial and stromal components. in general, breast tumors are highly hormonally dependent and growth hormone by its physiology may have a possible oncogenic potential. Therefore, the aim of this study was to determine the expression of growth hormone and growth hormone receptor in epithelial and stromal components of fibroadenomas. study group included 30 randomly chosen fibroadenomas from female patients aged between 18 and 69 years. The expression of growth hormone and growth hormone receptor was defined in both histologic components of fibroadenomas. growth hormone was expressed in 96.7% of both epithelial and stromal components of fibroadenomas, with stronger expression in the stromal component. The same percentage of positive reaction (96.7%) was obtained in the epithelial component of fibroadenomas for growth hormone receptor expression. only 6.7% of stromal components tested for growth hormone receptor were positive. The high expression of growth hormone and growth hormone receptor in fibroadenoma tissue indicates their possible role in the pathogenesis of this tumor. follow up of patients with high expression of growth hormone and growth hormone receptor may be suggested.Key words: Fibroadenoma; Growth hormone; Growth hormone receptorCorrespondence to: Božo Krušlin, MD, PhD, department of Pat-hology, school of medicine, University of Zagreb, Šalata 10, hr-10000 Zagreb, CroatiaE-mail: kruslin@mef.hrreceived september 20, 2012, accepted may 5, 2013Introductionfibroadenoma is one of the most common causes of a benign lump in the breast1. it may occur at any age after puberty but is most frequent in the third de-cade1. fibroadenoma presents as a painless, solitary, firm, slowly growing, mobile, well-defined nodule and less frequently it may appear in the form of mul-tiple nodules. The admixture of stromal and epithelial proliferation gives rise to two distinct growth patterns of no clinical significance2. both epithelial and strom-al components can show a wide spectrum of changes typically occurring at certain age, such as apocrine and squamous metaplasia in the epithelial component or focal and diffuse hypercellularity, extensive myx-oid change and hyalinization with dystrophic calci-fication, or ossification in the stromal component2. ductules vary in configuration; two classic patterns are described, intracanalicular when ductules are compressed by the stroma into clefts, and pericanali-cular when the stroma appears to surround ductules in a circumferential fashion1.Pituitary growth hormone (gh) acts on growth mainly through the regulation of the insulin-like growth factor (igf) system3. it binds with the growth hormone receptor (ghr), thus inducing the produc-tion of igfs (igf-i and igf-ii) in the majority of 236 Acta Clin Croat,  Vol. 52,   No. 2,  2013tanja Leniček et al. Expression of growth hormone and growth hormone receptor in fibroadenomas of the breastorgans and body tissues. These have autocrine, para-crine and endocrine actions on metabolism and cell proliferation, growth and differentiation3. The evidence that gh, igf-i and insulin can pro-mote and contribute to cancer progression comes from various sources, including transgenic and knockout mouse models and animal and human cell lines derived from cancers4. Cullen et al.5 report that igfs are mito-gens for many cancer cell types. in breast cancer cells, igf-i and igf-ii have both been shown to stimulate cell proliferation5. increased ghr level was found in colorectal carcinoma and other tumors including breast, squamous cell and basal cell carcinoma of the skin and melanoma but also preneoplastic lesions6-8. There are also epidemiologic data supporting a link between gh/igf status and cancer risk. Epide-miologic studies of patients with acromegaly showed an increased risk of colorectal cancer, although the risk of other cancers is unproven. Also, a long-term follow-up study of children deficient in gh treated with pituitary-derived gh has shown an increased risk of colorectal cancer9. The risk of prostate cancer is increased in men with elevated plasma igf-i, and this association is particularly strong in younger men, sug-gesting that early pathogenesis of prostate cancer may be connected with circulating igf-i plasma levels10. The aim of this study was to determine the expres-sion of gh and ghr in the epithelial and stromal components of fibroadenomas and to compare the re-sults both between these two compartments and be-tween fibroadenomas as a whole.Materials and MethodsThe study was performed on archival material taken from the Ljudevit Jurak department of Pathology, ses-tre milosrdnice University hospital Center. study group included 30 randomly chosen fibroadenomas from fe-male patients aged between 18 and 69 years. specimens were fixed in 10% buffered formalin, embedded in par-affin, cut at 5-μm thickness, and routinely stained with hematoxylin and eosin. The diagnosis of fibroadenoma was histologically confirmed in all cases. immunohistochemical staining for gh (dAKo, denmark, dilution 1:400) and ghr (novocastra, United Kingdom, dilution 1:100) was performed fol-lowing LsAb visualization method on dakotechmate tm automated machine for immunohistochemical staining with streptavidin immunoperoxidase (msiP protocol). The expression of gh and ghr was evalu-ated in both epithelial and stromal components of fi-broadenomas. The area with the highest number of gh/ghr positive cells (‘hot-spot’) was selected un-der low magnification field (x40), and then the reac-tion was evaluated under high magnification (x400) in 1000 tumor cells.The results of immunohistochemical staining were expressed semiquantitatively using the immunohis-tochemical staining index (isi), obtained by multiply-ing the intensity of the staining reaction (ir) and the percentage of immunoreactive cells (PrC).The intensity of the staining reaction was pre-sented as follows: 0 for negative reaction; 1 for weakly positive reaction; 2 for moderately positive reaction; and 3 for very strong reaction. The per-centage of immunoreactive cells was presented as follows: 0 for negative reaction; 1 for up to 20% of positive cells; 2 for more than 20% but less than 50% of positive cells; and 3 for over 50% of positive cells. According to the above mentioned conditions, four groups were established: isi 0 – negative reaction; isi 1 – weakly positive reaction (isi product 1-3); isi 2 – moderately positive reaction (isi product 4-6); and isi 3 – very strong reaction (isi product 7-9).Resultsstudy results are presented in table 1. in the epi-thelial component of fibroadenoma, gh was expressed in 29 of 30 samples (96.7%), with 7 samples showing Table 1. Immunohistochemical staining index (ISI) of growth hormone (GH) and growth hormone receptor (GHR) in epithelial and stromal components of fibroad-enomagh, epitheliumgh, stromaghr, epitheliumghr, stromaisi 0isi 1isi 2isi 3total1913730109203017148302820030isi 0 = negative reaction; isi 1 = weakly positive reaction; isi 2 = moderately positive reaction; isi 3 = very strong reactionActa Clin Croat,  Vol. 52,  No. 2,  2013 237tanja Leniček et al. Expression of growth hormone and growth hormone receptor in fibroadenomas of the breastvery strong reaction, 13 samples moderately positive and 9 samples weakly positive reaction. in the stromal component of fibroadenoma, gh was expressed in 29 of 30 samples (96.7%), with 20 samples showing very strong reaction and 9 samples moderately posi-tive reaction (fig. 1). in the epithelial component of fibroadenoma, ghr was expressed in 29 (96.7%) of 30 samples, with 8 samples showing very strong reac-tion, 14 samples showing moderately positive reaction and 7 samples showing weakly positive reaction (fig. 2). in the stromal component of fibroadenoma, ghr was practically not detected, with only 2 (6.7%) sam-ples showing a weakly positive reaction. All positive reactions were observed in the cytoplasm of epithelial and stromal cells.DiscussionLike normal breast tissue, benign proliferations developing in breast are under a complex system of controls by both systemic hormonal and local factors11. Although there are several studies that investigated the expression of gh and ghr in fibroadenomas, none of them included both of these markers. it is likely that gh plays an important role in the devel-opment of fibroadenomas since a number of studies have demonstrated an association between the soma-totropic axis and the risk of developing tumors of the mammary gland5,6.some investigators focused on the local produc-tion of gh. in the study by raccurt et al.12, in situ rt-PCr was used to detect gh mrnA in the tu-moral and non-tumoral epithelial components and also in the cells of reactive stroma. The intensity of cellular labeling was always stronger compared with that in the adjacent normal tissue irrespective of the component observed12. gh protein was also present in a significantly higher concentration in proliferative disorders of the mammary gland compared with nor-mal mammary gland12. our study confirmed the high presence of gh in fibroadenomas. gil-Puig et al.13 found the expression of Pit-1 mrnA and protein in both normal and tumorous hu-man breast. The transcription factor pituitary-1 (Pit-1) is usually mainly expressed in the pituitary gland, where it has a critical role in cell differentiation and is a transcriptional factor for gh and prolactin (PrL). The Pit-1 mrnA levels were found to be significantly increased in breast carcinoma compared with normal breast13. The increase in the number of cancer cells in breast as a consequence of autocrine production of gh is a result of both increased mitogenesis and decreased apoptosis14. mol et al.15 have come to a conclusion that the gene encoding gh is expressed in the mam-mary gland of a variety of species, including human, and that this appears to represent a contribution to the molecular explanation of the role of progestins on proliferation of mammary epithelium. briefly, there Fig. 2. Immunohistochemical staining showing positive reaction for growth hormone receptor in the cytoplasm of epithelial component of fibroadenoma. (X100)Fig. 1. Immunohistochemical staining, showing positive reaction for growth hormone in the cytoplasm of both epi-thelial and stromal components of fibroadenoma. (X200)238 Acta Clin Croat,  Vol. 52,   No. 2,  2013tanja Leniček et al. Expression of growth hormone and growth hormone receptor in fibroadenomas of the breastis evidence that locally produced gh is involved in neoplastic changes in the breast. The study by Kaulsay et al.16 showed that the effects of autocrine gh on hu-man mammary carcinoma cell behavior are mediated via the ghr. mertani et al.17 found the expression of ghr and prolactin receptor (PrLr) genes and their translation in epithelial cells of normal, proliferative and neoplas-tic lesions of the breast including fibroadenoma, where ghr was detected using immunohistochemistry both in the epithelial and stromal components. stromal cells were found sometimes positive, but the label-ing intensity was always weaker than for neoplastic epithelial cells, including fibroadenomas17. This corre-lates with our results where only 2 stromal samples of fibroadenoma tissue were positive for ghr compared to 29 positive epithelial samples. gebre-medhin et al.6 demonstrated greater ghr expression in breast carcinoma than in normal breast tissue. detecting the ghr by PCr, 75% of the samples (36 of 48) were evidently positive6. some studies were focused on circulating gh and/or igf in order to unfold the pathogenesis of breast tumors. hankinson et al.18 and toniolo et al.19 found a positive correlation between circulating igf-i concentration and the risk of breast cancer among premenopausal but not postmenopausal women. on the contrary, literature data show no statistically sig-nificant differences in the expression of estrogen and progesterone receptors in invasive ductal breast car-cinoma between premenopausal and postmenopausal women20. The results obtained by sawyer et al.21 re-vealed igf-i and igf-ii overexpression in fibroade-nomas and the most similar phylloides tumors, which seems to be important in their pathogenesis. in conclusion, it is obvious that there is stronger ex-pression of gh and ghr in fibroadenomas and other tumorous breast tissues than in normal breast tissue, which was confirmed in this study. our results show that there is a higher gh expression in the stromal than in the epithelial component of fibroadenoma. Likewise, ghr is mostly expressed in the epithelial component, while there is almost no expression in the stromal component, rendering epithelial component the probable main pathway for gh influence.based on our and previous studies, it can be con-cluded that the pathogenesis of breast tumors, such as fibroadenoma, is highly hormone dependent. follow up of patients with high expression of gh and ghr may be suggested. further research in this area may hopefully lead us to more efficient hormonally depen-dent therapy.AcknowledgmentThis work was supported in part by the ministry of science, Education and sports, republic of Croatia, project number 108-1081870-1884.References  1. ELLis io, PindEr sE, LEE Ahs. tumors of the breast. in: fLEtChEr Cdm, editor. diagnostic histopathol-ogy of tumors, 3rd edn. Philadelphia: Churchill Livingstone, 2007;906-7.  2. tAn Ph, tsE g, LEE A, simPson Jf, hAnbY Am. fibroepithelial tumors. in: LAKhAni sr, ELLis io, sChnitt sJ, tAn Ph, van de ViJVEr mJ, editors. who classification of tumours of the breast, 4th edn. Lyon: international Agency for research on Cancer, 2012;142-7.  3. mArtinELLi CE Jr, CUstodio rJ, AgUiAr-oL-iVEirA mh. Physiology of the gh-igf axis. Arq bras Endocrinol metab 2008;52:717-25.  4. CLAYton PE, bAnErJEE i, mUrrAY Pg, rEnE-hAn Ag. growth hormone, the insulin-like growth factor axis, insulin and cancer risk. nat rev Endocrinol 2011;7:11-24.  5. CULLEn KJ, ALLison A, mArtirE i, ELLis m, singEr C. insulin-like growth factor expression in breast cancer epithelium and stroma. breast Cancer res treat 1992;22:21-9.   6. gEbrE-mEdhin m, KindbLom Lg, wEnnbo h, tornELL J, mEis-KindbLom Jm. growth hormone receptor is expressed in human breast cancer. Am J Pathol 2001;158:1217-22.  7. JUKić Z, tomAs d, miJić A, KrUŠLin b. Expression of growth hormone receptor and growth hormone in colorec-tal carcinoma. hepatogastroenterology 2009;56:85-8.  8. ginArtE m, gArCiA-CAbALLEro t, fErnAn-dEZ-rEdondo V, bEirAs A, toribio J. Expression of growth hormone receptor in benign and malignant cutane-ous proliferative entities. J Cutan Pathol 2000;27:276-82.  9. JEnKins PJ, bEssEr m. Clinical perspective: acro-megaly and cancer: a problem. J Clin Endocrinol metab 2001;86:2935-41.10. stAttin P, bYLUnd A, rinALdi s, biEssY C, dEChAUd h, stEnmAn Uh, EgEVAd L, ribo-Li E, hALLmAns g, KAAKs r. Plasma insulin-like Acta Clin Croat,  Vol. 52,  No. 2,  2013 239tanja Leniček et al. Expression of growth hormone and growth hormone receptor in fibroadenomas of the breastgrowth factor-i, insulin-like growth factor-binding proteins, and prostate cancer risk: a prospective study. J natl Cancer inst 2000;92:1910-7.11. CoUrtiLLot C, PLU-bUrEAU g, binArt n, bAL-LEYgUiEr C, sigAL-ZAfrAni b, goffin V, KUt-tEnn f, KELLY PA, toUrAinE P. benign breast dis-eases. J mammary gland biol neoplasia 2005;10:325-35.12. rACCUrt m, LobiE PE, moUdiLoU E, gArCiA-CAbALLEro t, frAPPArt L, morEL g, mEr-tAni hC. high stromal and epithelial human gh gene expression is associated with proliferative disorders of the mammary gland. J Endocrinol 2001;175:307-18.13. giL-PUig C, sEoAnE s, bLAnCo m, mACiA m, gArCiA-CAbALLEro t, sEgUrA C, PErEZ-fEr-nAndEZ r. Pit-1 is expressed in normal and tumorous hu-man breast and regulates gh secretion and cell proliferation. Eur J Endocrinol 2005;153:335-44.14. KAULsAY KK, mErtAni hC, tornELL J, morEL g, LEE Ko, LobiE PE. Autocrine stimulation of human mammary carcinoma cell proliferation by human growth hor-mone. 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Lancet 1998;351:1393-406.19. tonioLo P, brUning Pf, AKhmEdKhA-noV A, bonfrEr Jm, KoEnig KL, LUKAnoVA A, shorE rE, ZELEniUCh-JACqUottE A. se-rum insulin-like growth factor-i and breast cancer. int J Can-cer 2000;88:828-32.20. PEtričEVić J, PEtKoVić m, JonJić n. Expression of estrogen and progesterone receptors in human ductal invasive breast carcinoma not otherwise specified: is there any differ-ence between premenopausal and postmenopausal women? Acta Clin Croat 2011;50:169-75.21. sAwYEr EJ, hAnbY Am, PoULsom r, JEffErY r, giLLEtt CE, ELLis io, ELLis P, tomLinson iP. beta-catenin abnormalities and associated insulin-like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast. J Pathol 2003;200:627-32.sažetakEKsPrEsiJA hormonA rAstA i rECEPtorA hormonA rAstA Kod fibroAdEnomA doJKET. Leniček, D. Kasumović, E. Štajduhar, T. Džombeta, Z. Jukić  i B. Krušlinfibroadenom je najčešća dobroćudna novotvorina dojke, a sastoji se od epitelne i stromalne komponente. Uzimajući u obzir hormonsku osjetljivost tumora dojke, te potencijalni onkogeni potencijal hormona rasta, cilj našega rada bio je utvrditi ekspresiju hormona rasta i receptora hormona rasta u epitelnoj i stromalnoj komponenti fibroadenoma. U studiju je bilo uključeno 30 nasumice odabranih fibroadenoma nastalih u žena u dobi od 18 do 69 godina. Ekspresija hormona rasta i receptora hormona rasta određivana je u obje histološke komponente fibroadenoma. Ekspresija hormona rasta nađena je u 96,7% epitelne i stromalne komponente, uz jaču izraženost ekspresije u stromalnoj komponenti. Ekspresija receptora hor-mona rasta u epitelnoj je komponenti također iznosila 96,7%, a u stromalnoj samo 6,7%. Visoka ekspresija hormona rasta i receptora hormona rasta u fibroadenomima označava potencijalnu ulogu ovoga hormona u patogenezi ispitivane bolesti, te bi se u bolesnica ove skupine mogle preporučiti češće kliničke kontrole. Ključne riječi: Fibroadenom; Hormon rasta; Receptor hormona rasta 

Ekspresija hormona rasta i receptora hormona rasta kod fibroadenoma dojke

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