Cui-Ge Yu*, Xiang-Yang Jiang, Bin Li, Lu Gan, Jian-Feng Huang

Transcription

1 DOI: Expressio of ER, R, C-erbB-2 ad Ki- i Edometrial Carcioma ad their Relatioships with the Cliicopathological Features RESEARCH ARTICLE Expressio of ER, R, C-erbB-2 ad Ki- i Edometrial Carcioma ad their Relatioships with the Cliicopathological Features Cui-Ge Yu*, Xiag-Yag Jiag, Bi Li, Lu Ga, Jia-Feg Huag Abstract Backgroud: To aalyze the expressio of estroge receptors (ER), progesteroe receptors (R), C-erbB-2 ad Ki- i edometrial carcioma (EC) ad their relatioships with the cliicopathological features. Materials ad Methods: Sixty-seve EC samples, ormal edometrial samples ad atypical hyperplasia edometrial samples were all selected i Shaaxi rovicial eople s Hospital from Ju., 2012 to Ju., The expressio of ER, R, C-erbB-2 ad Ki- i, ormal edometrial tissue ad atypical hyperplasia edometrial tissue was respectively detected usig immuohistochemical S method. The relatioships betwee the expressio of ER, R, C-erbB-2 ad Ki- ad the patiets cliicopathological features as well as their correlatios i were also aalyzed. Results: The positive expressio rates of ER ad R i were 44.8% ad 41.8%, respectively, dramatically lower tha i atypical hyperplasia edometrial tissue ad ormal edometrial tissue (<0.01). The positive expressio rates of C-erbB-2 ad Ki- i were 80.6% ad 64.2%, respectively, sigificatly higher tha i atypical hyperplasia edometrial tissue ad ormal edometrial tissue (<0.01). I, the expressio of ER ad R was closely associated with the differetiated degrees ad depth of myometrial ivasio (<0.05), while that of C-erbB-2 ad Ki- with the cliical stagig, differetiated degrees, depth of myometrial ivasio ad presece or absece of lymph ode metastasis (<0.05). Spearma correlatio aalysis further displayed that the expressio of ER was positively correlated with R (r=0.393, =0.001), but egatively with C-erbB-2 ad Ki- (r=-0.469, =0.000; r=-0.329, =0.007); The expressio of R was egatively correlated with C-erbB-2 ad Ki- (r=-0.273, =0.025; r=-0.251, =0.041), but that of C-erbB-2 positively with Ki- (r=0.342, =0.005). Coclusios: Abormal expressio of ER, R, C-erbB2 ad Ki- might play a importat role i edometrial maligat trasformatio ad cell differetiatio, so their joit detectio is likely to be a comprehesive combiatio of immue factors, which is of great importace for EC progosis. Keywords: Estroge receptors - progesteroe receptors - C-erbB-2 - Ki- - edometrial carcioma Asia ac J Cacer rev, 16 (15), Itroductio Edometrial carcioma (EC), origiatig from maligat epithelial tumors, is the most commo maligacy of the female geital tract i developed coutries (Kajo et al., 2015). It ca cause about deaths aually aroud the world ad is classified ito several histological subtypes icludig edometrioid ad serous histologies (Hog et al., 2015). I recet years, the icidece of EC has bee o a progressive rise due to extesive applicatio of hormoe-replacemet therapy i cliic, especially for the youg people. The occurrece of EC is ot oly related to the levels of estroge receptors (ER) ad progesteroe receptors (R), but also to the abormal activatio of proto-ocogees ad abormal proliferatio of cells (Srijaipracharoe et al., 2010; Bider et al., 2014). Tumorigeesis is the lesio caused by activatio of ocogees or iactivatio of tumor suppressor gees. EC pertais to hormoe-depedet tumors, ad both ER ad R i its tumor tissue are of great importace for the progosis ad cliical selectio of edocrie therapy. C-erbB-2 that ca ihibit tyrosiase activity plays a pivotal role i the cell growth, proliferatio ad differetiatio, ad its overexpressio is positively correlated with the maligat degrees of the tumor (Uharcek, 2008). As oe of the extesively-applied cell proliferatio markers, Ki- ca reflect the proliferatio degree of maligat cells ad is associated with the progressio, metastasis ad progosis of various maligacies. A lot of studies have show that that the progosis of patiets with EC is closely related to the age, histological grade, depth of myometrial ivasio ad/or cervical ivasio, ad the presece of lymph ode metastases (Faria et al., 2015; Takahashi et al., 2015), ad some biological idicators icludig hormoe receptors, ocogees ad tumor suppressor gees are also Departmet of Gyecology ad Obstetrics, Shaaxi rovicial eople s Hospital, Xi a, Shaaxi, Chia *For correspodece: ycg925@sia.cm. Asia acific Joural of Cacer revetio, Vol 16,

2 Cui-Ge Yu et al ivolved (Ito et al., 1997), but these idicators are short of comprehesive verificatio of cliical trials with large sample sizes. Hece, i this study, the expressio of ER, R, C-erbB-2 ad Ki- i was detected usig immuohistochemical S method ad their relatioships with the cliicopathological features of EC were also ivestigated so as to provide valuable idicators for the cliical treatmet ad progostic evaluatio of EC. Materials ad Methods Sample source Sixty-seve EC samples archived i Departmet of athology ad cofirmed by two pathologists i Shaaxi rovicial eople s Hospital were selected from Ju., 2012 to Ju., They were at the age of 29-68, with the mea age of Amog patiets with EC, there were 22 cases with high differetiatio, 19 with moderate differetiatio ad 26 with poor differetiatio; Accordig to the depth of myometrial ivasio, the ivasive depth was less tha 1/2 i 39 cases, equal to or more tha 1/2 i 28 cases; 29 cases suffered from lymph ode metastasis, while 38 ot; Based o the cliical stagig, there were 22 cases i phase I, 16 i phase II ad 29 i phase III-IV. Besides, the edometrial samples with other gyecological diseases that uderwet surgical resectio or uterie curettage were also selected at the same term, i which ormal edometrial samples ad atypical hyperplasia edometrial samples were both cases. All the erolled samples had o history of radiotherapy, chemotherapy, ER ad R admiistratio, ad the cliicopathological files were complete. This research was approved by Ethics Committee of Shaaxi rovicial eople s Hospital, ad the patiets or their relatives siged the iformed coset form. Major reagets Mouse ati-huma ER, R, C-erbB-2 ad Ki- moocloal atibodies were all purchased from Fujia Maixi Biotech Co., Ltd. Ezyme-labeled goat ati-mouse IgG polymer as the secodary atibody ad ready-to-use DAB coloratio kit were provided by Beijig Zhogsha Golde Bridge Biotech Co., Ltd. Detectio method All the samples were fixed by paraformaldehyde, embedded by paraffi ad cotiuously cut ito slices with the thickess of 4 μm. The expressio of ER, R, C-erbB-2 ad Ki- i, atypical hyperplasia edometrial tissue ad ormal edometrial tissue was all detected usig immuohistochemical S method. Specific steps were as follows: (1) bake the sectios at 68 for 20 mi; (2) carry out dewaxig ad dehydratio respectively with xylee ad gradiet alcohol, ad the apply 3% H 2 O 2 to icubate 10 mi at 37 ad BS to swash 3 times, 5 mi per time; (3) coduct atige repair i citrate for mi, ad the apply BS to swash 3 times, 5 mi per time after coolig to room temperature; (4) use the ormal goat serum workig solutio for sealig 10 mi at 37, dropwise add primary atibody, stay overight for icubatio at 4, ad the coduct secodary atibody icubatio 90 Asia acific Joural of Cacer revetio, Vol 16, 2015 after applyig BS to swash 3 times; (5) apply DAB for coloratio, hematoxyli for couterstaiig, xylee for trasparecy ad eural resi for sealig sectios. All the operatios were carried out strictly accordig to the kit istructios. Kow positive sectios of EC were regarded as positive cotrols ad BS as egative cotrols istead of primary atibodies. Judgig criteria All the sectios were observed ad judged idepedetly by two pathologists uder the coditio of ukow cliical data. The positive expressio of ER ad R was maily located i cell uclei, maifestig differet sizes of brow-yellow graules; The positive expressio of C-erbB-2 was primarily located i cytomembrae; The positive expressio of Ki- was defied as presece of brow-yellow graules i cell uclei or i both cell uclei ad cytomembrae. The results were evaluated usig semi-quatitative scorig method: (1) Five high-power visual fields ( 400) i each sectio were radomly selected to calculate the total umber of tumor cells ad umber of positive cells. The proportios of positive cells 5%, beig 6%-25% 26%-50%, 51%-75% ad >75% were respectively scored 0, 1, 2, 3 ad 4 poits; (2) The scores were made accordig to staiig itesity, amely colorless, light yellow, brow yellow ad sepia were represeted by 0, 1, 2, ad 3 poits, respectively. The product of two scores above was as the fial score, amely 0 poit beig egative (-), 1-4 poits beig weakly positive (+), 5-8 poits beig positive (++) ad 9-12 poits beig strogly positive (+++). Statistical data aalysis SSS 15.0 software package was used for statistical aalysis. The eumeratio data were compared usig x 2 test ad expressed by percetages. Relatioships betwee the expressio of ER, R, C-erbB-2 ad Ki- i ad the cliicopathological features were aalyzed by x 2 test of the four-fold table, ad their correlatios i EC tissue were aalyzed by Spearma correlatio aalysis. All statistical tests were performed i a two-sided way. <0.05 was cosidered to be statistically sigificat. Results Expressio of ER, R, C-erbB-2 ad Ki- i differet edometrial tissues The positive expressio of ER ad R was maily located i cell uclei, maifestig differet sizes of brow-yellow graules (Figure 1; Figure 2). The positive expressio of C-erbB-2 was primarily located i cytomembrae (Figure 3). The positive expressio of Ki- was defied as presece of brow-yellow graules i cell uclei or i both cell uclei ad cytomembrae (Figure 4). As show i Table 1 ad Table 2, the positive expressio rates of ER ad R i were 44.8% ad 41.8%, respectively, dramatically lower tha i atypical hyperplasia edometrial tissue (79.2%, ER =0.000; 77.4%, R =0.000) ad ormal edometrial tissue (90.6%, ER =0.000; 92.5%, R =0.000). Sigificat differece was

3 DOI: Expressio of ER, R, C-erbB-2 ad Ki- i Edometrial Carcioma ad their Relatioships with the Cliicopathological Features Figure 1. A. ositive Expressio of ER i Normal Edometrial Tissue ( 400); B. ositive Expressio of ER i Atypical Hyperplasia Edometrial Tissue ( 400); C: ositive Expressio of ER i ( 400) Figure 2. A. ositive Expressio of R i Normal Edometrial Tissue ( 400); B. ositive Expressio of R i Atypical Hyperplasia Edometrial Tissue ( 400); C. ositive Expressio of R i ( 400) Figure 3. A. ositive Expressio of C-erbB-2 i Normal Edometrial Tissue ( 400); B. ositive Expressio of C-erbB-2 i Atypical Hyperplasia Edometrial Tissue ( 400); C. ositive Expressio of C-erbB-2 i ( 400) Figure 4. A. ositive Expressio of Ki- i Normal Edometrial Tissue ( 400); B. ositive Expressio of Ki- i Atypical Hyperplasia Edometrial Tissue ( 400); C. ositive Expressio of R i Ki- tissue ( 400) Table 1. Compariso o ER Expressio i Differet Edometrial Tissues [(%)] Atypical hyperplasia edometrial tissue ER ositive expressio Negative expressio 37(55.2) 11(20.8) 5(9.4) 30(44.8) (79.2) 48(90.6) Table 2. Compariso o R Expressio i Differet Edometrial Tissues [(%)] Atypical hyperplasia edometrial tissue R ositive expressio 100.0expressio Negative 39(58.2) 12(22.6) 4(7.5) (41.8) (77.4) 49(92.5) Table 3. Compariso o C-erbB-2 Expressio i Differet Edometrial Tissues[(%)] 13(19.4) 25(47.2) 25.0 (100.0) (80.6) 28(52.8) (0.0) Table 4. Compariso o Ki- Expressio i Differet Edometrial Tissues[(%)] 0 24(35.8) 35(66.0) 50(94.3) also preseted by compariso to the positive expressio rates of ER ad R i atypical hyperplasia edometrial tissue ad ormal edometrial tissue (=2.650, =0.104; =4.711, =0.030). As show i Table 3 ad Table 4, the positive Newly diagosed with treatmet Atypical hyperplasia edometrial tissue Negative expressio (64.2) 18(34.0) 3(5.7) expressio rates of C-erbB-2 ad Ki- i were 80.6% ad 64.2%, respectively, sigificatly higher tha i atypical hyperplasia edometrial tissue (52.8%, C=0.001; 34.0%, Ki-=0.001) ad ormal edometrial erbb-2 tissue (0.0%, C-erbB-2=0.000; 5.7%, Ki-=0.000). There Asia acific Joural of Cacer revetio, Vol 16, Ki- ositive expressio Newly diagosed without treatmet Remissio Atypical hyperplasia edometrial tissue 54.2 C-erbB Negative expressio ositive expressio 50.0 ersistece or recurrece Noe 31 3

4 Cui-Ge Yu et al Table 6. Relatioships Betwee C-erbB-2, Ki- Expressio ad the atiets Cliicopathological Features[(%)] Cliicopathological Features C-erbB-2 Expressio x 2 Ki- Expressio x 2 Negative ositive Negative ositive Cliical stagig I-II 38 12(31.6) 26(68.4) (50.0) 19(50.0) III-IV 29 1(3.4) 28(96.6) 5(17.2) 24(82.8) Differetiated degrees High differetiatio 22 8(36.4) 14(63.6) (59.1) 9(40.9) Moderate differetiatio 19 3(15.8) 16(84.2) 5(26.3) 14(73.7) oor differetiatio 26 2(7.7) 24(92.3) 6(23.1) 20(76.9) Depth of myometrial ivasio <1/ (30.8) 27(69.2) (46.2) 21(.8) /2 28 1(3.6) 27(96.4) 6(21.4) 22(78.6) Lymph ode metastasis Yes 29 2(6.7) 28(93.3) (20.7) 23(79.3) No 38 12(31.6) 26(68.4) 18(47.4) 20(52.6) Table 5. Relatioships Betwee ER, R Expressio ad the atiets Cliicopathological Features[(%)] Cliicopathological Features ER Expressio x 2 R Expressio x 2 Negative ositive Negative ositive Cliical stagig I-II 38 18(47.4) 20(52.6) (50.0) 19(50.0) III-IV 29 19(65.5) 10(34.5) 18(62.1) 11(37.9) Differetiated degrees High differetiatio 22 9(40.9) 13(59.1) (36.4) 14(63.6) Moderate differetiatio 19 8(42.1) 11(57.9) 8(42.1) 11(57.9) oor differetiatio 26 20(76.9) 6(23.1) 21(80.8) 5(19.2) Depth of myometrial ivasio <1/ (41.0) 23(59.0) (38.5) 24(61.5) / (75.0) 7(25.0) 22(78.6) 6(21.4) Lymph ode metastasis Yes 29 18(62.1) 11(37.9) (65.5) 10(34.5) No 38 19(50.0) 19(50.0) 18(47.4) 20(52.6) was statistical sigificace by compariso to the positive expressio rates of C-erbB-2 ad Ki- i atypical hyperplasia edometrial tissue ad ormal edometrial tissue (x 2 =38.051, =0.000; x 2 =13.361, =0.000). Relatioships betwee the expressio of ER, R, C-erbB-2 ad Ki- ad the patiets cliicopathological features I, the expressio of ER ad R was closely associated with the differetiated degrees ad depth of myometrial ivasio ( ER =0.017, 0.006; R =0.003, 0.001) istead of the cliical stagig ad presece or absece of lymph ode metastasis (>0.05) (Table 5). The expressio of both C-erbB-2 ad Ki- was itimately related to the cliical stagig, differetiated degrees, depth of myometrial ivasio ad presece or absece of lymph ode metastasis ( C-erbB-2 =0.004, 0.039, 0.005, 0.012; Ki- =0.006, 0.021, 0.037, 0.024) (Table 6). Correlatio amog the expressio of ER, R, C-erbB-2 ad Ki- i EC I, the expressio of ER was positively correlated with R (r=0.393, =0.001), but egatively with C-erbB-2 ad Ki- (r=-0.469, =0.000; r=-0.329, =0.007); The expressio of R was egatively correlatio with C-erbB-2 ad Ki- (r=-0.273, =0.025; r=-0.251, =0.041); The expressio of C-erbB-2 had a sigificatly positive correlatio with Ki- (r=0.342, =0.005). Discussio EC, a primary epithelial tumor, is usually accompaied by glad differetiatio ad has the potecy of ifiltratio ito uterie walls ad distat dissemiatio. I recet years, the icidece of EC is o the rise due to the extesio of average life i the populatio ad applicatio of hormoe-replacemet therapy durig meopause. The uterus i female geital system is a target orga of sexual hormoe, where the tumors are regarded as the sexual hormoe-depedat tumor. Extesively distributed i the histocytes of female reproductive orgas, both ER ad R are essetial i the systemic regulatio of estroge ad progestoge i female reproductive orgas. Their productio ad fuctios ca be affected whe ormal histocytes suffer from variatio. Nevertheless, the therapeutic respose of EC to hormoe depeds o the level of hormoe receptors i the tumor tissue. High level of receptors has better respose to edocriotherapy (Kuramoto et al., 1989). This study applied immuohistochemical S method to detect the expressio of ER ad R i three differet edometrial tissues, ad the results showed that the positive expressio of ER ad R i was markedly lower tha 92 Asia acific Joural of Cacer revetio, Vol 16, 2015

5 DOI: Expressio of ER, R, C-erbB-2 ad Ki- i Edometrial Carcioma ad their Relatioships with the Cliicopathological Features i atypical hyperplasia edometrial tissue ad ormal edometrial tissue, ad that i ormal edometrial tissue was the lowest. Additioally, with the icrease of tumor differetiated grades ad myometrial ivasive depth, the positive expressio rates of ER ad R wet dow, which might be related to icreased maligat degrees of tumor cells, elevated DNA cotet i cacer cell uclei ad chage of ormal ezyme activity that all impact the sythesis of ER ad R. C-erbB-2 is a chief proto-ocogee of EC, which plays a pivotal role i its biological behavior (Zhao et al., 2009). Its gee amplificatio ad protei overexpressio have bee associated with poor progosis i several solid tumors, icludig breast ad gastric cacer (Thompso et al., 2011; Sharifah et al., 2008). C-erbB-2 is ot expressed or lowly expressed i ormal edometrial tissue, but its expressio rate goes up gradually from atypical edometrial hyperplasia to EC (Ioachi 2005). I this study, the positive expressio rate of C-erbB-2 i was 80.6%, sigificatly higher tha i ormal edometrial tissue (0.0%) ad atypical hyperplasia edometrial tissue (2.8%), coformig to the above reports. Amplificatio ad overexpressio of C-erbB-2 gee ca cause the maligat trasformatio of ormal cells. The ivasio of highly-expressed tumors is higher, ad progosis is poorer. There are a lot of research results regardig the relatioships betwee amplificatio or overexpressio of C-erbB-2 gee ad EC biological behaviors. Morriso et al. aalyzed 483 patiets with EC ad foud that C-erbB-2 overexpressio was related to histological grades (Morriso et al., 2006). However, Seki et al. believed that the positive expressio of C-erbB-2 was ot associated with histological grades or FIGO stagig (Seki et al., 1998). The research results i this study displayed that the positive expressio of C-erbB-2 i phase III-IV patiets was higher tha i phase I-II, showig that C-erbB-2 expressio icreases with the cliical stagig elevatig; As the tumor differetiated grades icreased, C-erbB-2 expressio wet up obviously, idicatig that C-erbB-2 expressio is related to histological differetiatio. The higher the positive expressio of C-erbB-2 was, the poorer the histological differetiatio was; The positive expressio of C-erbB-2 i patiets with lymph ode metastasis or depth of myometrial ivasio 1/2 was also dramatically higher tha those without lymph ode metastasis or depth of myometrial ivasio <1/2, showig C-erbB-2 expressio icreases with aggravatio of lymph ode metastasis ad myometrial ivasio. All above results suggest that the tumors with high C-erbB-2 expressio are stroger i ivasio, rapid i disease progressio ad poor i progosis. The proliferatio of tumor cells depeds o their biological behaviors. The occurrece ad progressio of EC are related to various factors, but abormal cell proliferatio exerts a certai effect (Gurda et al., 2014). Ki- pertais to o-histoe proteis ad is a affirmative proliferatio marker at preset. I cell cycle, its expressio begis to appear i phase G 1, icreases i phases S ad G 2, reaches to the peak i phase M, ad disappears rapidly i the advaced stage of cell divisio, but it is ot expressed i phase G 0. Ki- becomes the most reliable idex i the detectio of tumor cell proliferatio activity due to short half-life period. I this study, the positive expressio of Ki- was 64.2%, dramatically higher tha i ormal edometrial tissue (5.7%) ad atypical hyperplasia edometrial tissue (34.0%), idicatig that Ki- expressio goes up successively i ormal edometrial tissue, atypical hyperplasia edometrial tissue ad, which may be related to the ehaced cell proliferatio. The expressio of Ki- i was itimately related to the cliical stagig, differetiated degrees, depth of myometrial ivasio ad presece or absece of lymph ode metastasis, illustratig that Ki- expressio icreases with the elevatio of tumor maligat degrees; The stroger cell proliferatio is, the higher maligat degrees are. Hece, Ki- ca be a importat idex for evaluatig EC proliferatio ad differetiatio ad has key biological ad cliical sigificace i EC occurrece, progressio ad diagosis. Correlatio aalysis further revealed that the expressio of ER i was positively correlated with R, ad egatively with C-erbB2 ad Ki-, illustratig that C-erbB2 is expressed weakly i hormoe-depedat, while strogly i o-hormoe-depedat ; The lower the expressio rates of ER ad R are, the more sigificat cell proliferatio is. Besides, the expressio of C-erbB-2 had a sigificatly positive correlatio with Ki-. All these results suggest that abormal expressio of ER, R, C-erbB2 ad Ki- might play a importat role i edometrial maligat trasformatio ad cell differetiatio, so their joit detectio is likely to be a comprehesive combiatio of immue factors, which is of great importace for EC progosis. Refereces Bider S, Mutch DG (2014). Update o progostic markers for edometrial cacer. Womes Health (Lod Egl), 10, Faria SC, Sagebiel T, Balachadra A, et al (2015). Imagig i edometrial carcioma. Idia J Radiol Imagig, 25, Gurda GT, Baras AS, Kurma RJ (2014). Ki- idex as a acillary tool i the differetial diagosis of proliferative edometrial lesios with secretory chage. It J Gyecol athol, 33, Hog B, Le Gallo M, Bell DW (2015). The mutatioal ladscape of edometrial cacer. Curr Opi Geet Dev, 30, Ioachi E (2005). Immuohistochemical tumour markers i edometrial carcioma. Eur J Gyaecol Ocol, 26, Ito K, Sasao H, Yabuki N, et al (1997). Immuohistochemical study of Ki- ad DNA topoisomerase II i huma edometrium. 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6 Cui-Ge Yu et al Sharifah NA, Lee BR, Clarece-Ko CH, et al (2008). C-erbB-2 oco-protei expressio i breast cacer: relatioship to tumour characteristics ad short-term survival i Uiversiti Kebasaa Malaysia Medical Cetre. Asia ac J Cacer rev, 9, Srijaipracharoe S, Tagjitgamol S, Tavaich S, et al (2010). Expressio of ER, R, ad Her-2/eu i edometrial cacer: a cliicopathological study. Asia ac J Cacer rev, 11, Takahashi R, Mabuchi S, Kawao M, et al (2015). rogostic sigificace of systemic eutrophil ad leukocyte alteratios i surgically treated edometrial cacer patiets: a mooistitutioal study. Gyecol Ocol, 137, Thompso SK, Sulliva TR, Davies R, et al (2011). Her-2/eu gee amplificatio i esophageal adeocarcioma ad its ifluece o survival. A Surg Ocol, 18, Uharcek (2008). rogostic factors i edometrial carcioma. J Obstet Gyaecol Res, 34, Zhao FJ, Zhag SL, Ma L, et al (2009). Ihibitory effects of c-erbb-2 atisese oligoucleotide trasfectio o uterie edometrial cacer Ishikawa cell lies. Eur J Gyaecol Ocol, 30, Asia acific Joural of Cacer revetio, Vol 16, 2015