The objective of this study was to verify the frequency of P53 and BCL-2 immunohistochemical expression in 98 patients with endometrial carcinoma, and to correlate it with clinical stage and patient survival. A significant difference was found regarding the frequency of P53 expression when comparing type I and II tumors (23.7% and 54.5%, respectively; p = 0.006). A positive correlation was observed between P53 immunoexpression and patient survival in type I and II tumors (p = 0.009 and p = 0.036, respectively). BCL-2 expression was significantly more frequent in early clinical stages in both types of endometrial cancer (p &lt; 0.001 and 0.002) and correlated with a decrease in overall survival in type I endometrial cancer (p = 0.014). Thus, the prognostic value of these biomarkers in endometrial cancer needs to be further investigated. (&lt;i&gt;Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 631&amp;#8211;635&lt;/i&gt;

Bozena Dobrzycka

Jacek Niklinski

Lech Chyczewski

Magdalena Garbowicz

Marek Kulikowski

Slawomir J. Terlikowski

The objective of this study was to verify the frequency of P53 and BCL-2 immunohistochemical expression
in 98 patients with endometrial carcinoma, and to correlate it with clinical stage and patient survival.
A significant difference was found regarding the frequency of P53 expression when comparing type I and II tumors
(23.7% and 54.5%, respectively; p = 0.006). A positive correlation was observed between P53 immunoexpression
and patient survival in type I and II tumors (p = 0.009 and p = 0.036, respectively). BCL-2 expression was
significantly more frequent in early clinical stages in both types of endometrial cancer (p < 0.001 and 0.002) and
correlated with a decrease in overall survival in type I endometrial cancer (p = 0.014). Thus, the prognostic
value of these biomarkers in endometrial cancer needs to be further investigated. (Folia Histochemica et Cytobiologica
2011; Vol. 49, No. 4, pp. 631&#8211;635

Dobrzycka, Bozena

Terlikowski, Slawomir J.

Garbowicz, Magdalena

Niklinski, Jacek

Chyczewski, Lech

Kulikowski, Marek

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 49, No. 4, 2011pp. 631–635©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201110.5603/FHC.2011.0086www.fhc.viamedica.plORIGINAL STUDYCorrespondence address: B. Dobrzycka, Department ofObstetrics, Gynecology and Obstetrics/Gynecological Care,Medical University of Bialystok,Warszawska Str. 15, 15–062 Bialystok, Poland;tel.: (+ 48 85) 748 88 69, fax: (+ 48 85) 748 88 60;e-mail: bdobrzycka@gmail.comThe prognostic significance of theimmunohistochemical expression of P53 and BCL-2in endometrial cancerBozena Dobrzycka1, Slawomir J. Terlikowski1, Magdalena Garbowicz2,Jacek Niklinski2, Lech Chyczewski3, Marek Kulikowski41Department of Obstetrics, Gynecology and Obstetrics/Gynecological Care,Medical University of Bialystok, Poland2Department of Clinical Molecular Biology, Medical University of Bialystok, Poland3Department of Pathomorphology, Medical University of Bialystok, Poland4Department of Perinatology, Medical University of Bialystok, PolandAbstract: The objective of this study was to verify the frequency of P53 and BCL-2 immunohistochemical ex-pression in 98 patients with endometrial carcinoma, and to correlate it with clinical stage and patient survival.A significant difference was found regarding the frequency of P53 expression when comparing type I and II tumors(23.7% and 54.5%, respectively; p = 0.006). A positive correlation was observed between P53 immunoexpres-sion and patient survival in type I and II tumors (p = 0.009 and p = 0.036, respectively). BCL-2 expression wassignificantly more frequent in early clinical stages in both types of endometrial cancer (p < 0.001 and 0.002) andcorrelated with a decrease in overall survival in type I endometrial cancer (p = 0.014). Thus, the prognosticvalue of these biomarkers in endometrial cancer needs to be further investigated. (Folia Histochemica et Cytobi-ologica 2011; Vol. 49, No. 4, pp. 631–635)Key words: endometrial cancer, P53, BCL-2, prognosisIntroductionEndometrial carcinoma is one of the commonest gy-necological malignancies. The age-adjusted incidencerate in Poland is 13.7 per 100,000 women per year[1]. Acknowledged clinicopathological prognosticmarkers used to determine the course of endometri-al carcinomas include histological type, tumor grade,depth of myometrial invasion, lymph-node involve-ment and clinical stage [2]. In the majority of cases,the neoplasm is histologically diagnosed as an en-dometrial carcinoma of endometrioid type (type I)and its stage at the time of diagnosis as type I accord-ing to the criteria of the International Federation ofGynecology and Obstetrics (FIGO). The second ma-jor group of endometrial cancer is histologically clas-sified as serous carcinoma (type II) [2].In the past few years, several studies have assessedthe clinical relevance of different biological factorsevaluated in tissue samples from patients with en-dometrial cancer in order to detect markers capableof predicting the outcome [3–8]. Several publicationshave regarded the TP53 and the BCL-2 genes as pos-sible markers of endometrial cancer aggressiveness[9–11]. Some 20–50% of endometrial carcinoma cas-es show abnormal P53 expression. The expression ofP53, as seen in immunohistochemical analysis, seemsto be related to undifferentiated, aggressive tumors,with a poor prognosis [6]. BCL-2 expression is rela-632 B Dobrzycka et al.©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201110.5603/FHC.2011.0086www.fhc.viamedica.pltively high in the normal endometrium, decreases inendometrial cancer and has been related to well-dif-ferentiated endometrial tumors, early-stage diseaseand diseases with a good prognosis [9, 10].The aim of the present study was to estimate thefrequency of P53 and BCL-2 immunohistochemicalexpression in patients who have been surgically treat-ed for endometrial carcinoma, and to correlate thisexpression with clinical stage and survival.Material and methodsPatients and clinical samples. A total of 98 patients withendometrial cancers (aged 49–72, median value — 61.7years) treated at the Department of Gynecology MedicalUniversity of Bialystok and the Department of GynecologyDistrict Hospital in Bialystok between 1999 and 2003, wereincluded in this study. None of the patients had receivedchemotherapy, hormonal therapy or radiation therapy pri-or to surgery. All patients had primary cancers and werereceiving first treatment. Cases selected in the present studyshowed the same stage, both clinically and surgically. Alltumors were staged according to the FIGO criteria. All pa-tients underwent abdominal hysterectomy and bilateraloophorectomy. Pelvic and paraaortic lymphadenectomy wasdone at stage II (34 cases) and stage III (16 cases). Adju-vant chemotherapy was added to the treatment for stageIII patients.Clinicopathological information was obtained from med-ical charts. Histopathological examination was performedaccording to the WHO classification. Representative sam-ples of hysterectomy specimens were stained with H&E forlight microscopic study and evaluated to confirm a tumorstage and histological type. Patients were informed and gavetheir consent for the study. The protocol was previously ap-proved by the Bioethical Committee of the Medical Uni-versity of Bialystok. Follow-up data was completed untilJanuary 2010.Immunohistochemical analysis. The following primary an-tibodies were used: monoclonal mouse anti-human P53 pro-tein, Clone: Do-7; Code M7001 and monoclonal mouse anti-human BCL-2 oncoprotein, Clone: 124; Code M0887(Dako, Glostrup, Denmark). All reactions were performedusing positive controls (breast cancer for P53 expression andtonsils for BCL-2 expression).After deparaffinization and rehydration, antigen retriev-al, inactivation of endogenous peroxidase and blocking ofnon-specific reactions, the sections were incubated for 2 hat ambient temperature with a diluted solution of primaryantibodies (1:50 for P53 and 1:100 for BCL-2). Primaryantibody binding was revealed by streptavidin-biotin per-oxidase complex and diaminobenzidine tetrahydrochloride(LSAB, Dako, Glostrup, Denmark).Tumors were considered positive for P53 expression if10% or more of the nuclei of tumor cells stained brown,and for BCL-2 expression if 10% or more of the cytoplasmof tumor cells stained brown.Statistical analysis. Statistical analysis was performed us-ing Statistica software version 9.0PL (StatSoft, Inc., Stat-Soft Polska Sp. z o.o., Poland). A chi-square test was usedto evaluate the relationship between categorical variables.Fisher’s exact test was used to determine significance be-tween the two groups. A p-value of < 0.05 was consideredas statistically significant. In addition, survival time was cal-culated from the date of surgery to the date of death andsurvival analysis was performed using the Kaplan–Meiermethod.ResultsThe tumors were classified as follows: 76 cases weretype I (endometrioid endometrial carcinomas) and22 cases were type II (19 cases of serous and three ofclear-cell carcinomas). Among patients with type Iendometrial cancer, 43 had tumors classified as stage I,24 had tumors classified as stage II, and nine had tu-mors classified as stage III.The type II tumors were classified as follows: fivecases were in stage I, ten cases were in stage II, andseven cases were in stage III. All patients were fol-lowed up. At a median follow-up of 60 (range 1–126)months, 39 (39.8%) patients had died as a conse-quence of cancer progression.In type I tumors, P53 immunoexpression was pos-itive in 18 cases (23.7%), while BCL-2 immunoex-pression was positive in 44 cases (57.9%). In type IItumors, P53 and BCL-2 immunoexpression was de-tected in 12 cases (54.5%) and eight cases (36.4%),respectively. The correlations of P53 and BCL-2 im-munoexpression between these two types of tumorsare summarized in Table 1.No correlation was found between the frequencyof P53 immunoexpression in both types of tumors andstages (p = 0.17 and p = 0.084). P53 immunoexpres-sion was more frequent in type II tumors (p = 0.006).A statistically significant correlation was found be-tween the frequency of BCL-2 immunoexpression andTable 1. Comparison of P53 and BCL-2 immunoexpressionbetween patients with type I and II endometrial cancerNo. of cases P53 BCL-2Type I no. (%) 76 18 (23.7) 44 (57.9)Type II no. (%) 22 12 (54.5) 8 (36.4)p 0.006 0.041633Prognostic significance of P53 and BCL-2 in EC©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201110.5603/FHC.2011.0086www.fhc.viamedica.plthe clinical stage of type I and II tumors (p < 0.001and p = 0.002, respectively) (Table 2).Among patients with positive P53 immunoexpres-sion in type I endometrial cancer, the Kaplan–Meiersurvival estimates of the 1-year, 5-year, and 10-year sur-vival rates were 97.4%, 92.1%, and 90.8%, respectively,while the respective rates among patients with negativeP53 immunoexpression were 97.4%, 93.4%, and 93.4%.A statistically significant difference was observed be-tween survival rates over time (p = 0.009) (Figure 1).In the group with type II endometrial cancer withpositive P53 immunoexpression, the Kaplan–Meiersurvival estimates of the 1-year, 5-year, and 10-yearsurvival rates were 86.4%, 77.3%, and 72.7%, respec-tively, while the respective rates among endometrialTable 2. Comparison of P53 and BCL-2 immunoexpressionbetween patients with type I and type II endometrial cancerof different FIGO stageNo. of cases P53 no. (%) BCL-2 no. (%)Type IStageI 43 (56.6) 7 (16.3) 38 (88.4)II 24 (31.6) 6 (25.0) 5 (20.8)III 9 (11.8) 4 (44.4) 1 (11.1)p 0.17 < 0.001Type IIStageI 5 (22.7) 2 (40.0) 5 (100)II 10 (45.5) 8 (80.0) 3 (42.9)III 7 (31.8) 2 (28.6) 0p 0.084 0.002Figure 1. Kaplan–Meier survival analysis concerningP53 immunoexpression and cumulative survival in type Iendometrial cancerFigure 2. Kaplan–Meier survival analysis concerningP53 immunoexpression and cumulative survival in type IIendometrial cancerFigure 3. Kaplan–Meier survival analysis concerningBCL-2 immunoexpression and cumulative survival in type Iendometrial cancercancer patients with negative P53 immunoexpressionwere 100%, 86.4%, and 84.8%. A statistically signifi-cant difference was observed between survival ratesover time (p = 0.036) (Figure 2).In type I endometrial cancer patients with posi-tive BCL-2 immunoexpression, the 1-year, 5-year, and10-year survival rates were 96.1%, 94.7%, and 94.7%,compared to those with negative BCL-2 immunoex-pression (93.4%, 90.8%, and 89.5%, respectively), andthe differences in survival were significant (p = 0.014)(Figure 3).Among patients with positive BCL-2 immunoex-pression in type II endometrial cancer, the Kaplan–634 B Dobrzycka et al.©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201110.5603/FHC.2011.0086www.fhc.viamedica.pl–Meier survival estimates of the 1-year, 5-year, and10-year survival rates were 95.5%, 86.4%, and 86.4%,respectively, while the respective rates among patientswith negative BCL-2 immunoexpression were 90.9%,81.8%, and 63.6%. No statistically significant differ-ence was observed between survival rates over time(p = 0.737) (Figure 4). The cumulative disease-spe-cific survival curves of patients grouped according toP53 and BCL-2 immunoexpression are shown inFigures 1–4.DiscussionA recent model of endometrial carcinogenesis re-vealed that there are two different types of tumor.Type I tumors develop slowly and progressively asa result of endometrial hyperplasia in unopposed es-trogen activity. These tumors are characterized bysustained BCL-2 expression and late mutations to theTP53 gene, as confirmed by the low expression of P53in stages I and II in our study. On the other hand,type II tumors develop in an atrophic epithelium with-out estrogen stimulation. They are aggressive andoccur in a distinct biological environment. Gene mu-tation and P53 protein accumulation are often foundin type II lesions [10, 12, 13].The presence of P53 mutations in endometrialcancer has been extensively studied in literature. Ap-proximately one in three endometrial cancers haveabnormalities of P53 by immunohistochemistry [7, 14–17].A number of studies have shown that both P53 over-expression and mutation are associated with poorprognosis [9, 10, 14, 15, 17]. It is worth noting thatearlier grade 3 endometrioid carcinomas were classi-fied as type II tumors. Molecular data now exists sug-gesting that most grade 2, and some grade 3, tumorshave molecular profiles similar to grade 1 tumors [12].In type II endometrial cancer, approximately 80%of carcinomas have mutations in P53, implying a rolefor its inactivation early in the development of thisaggressive tumor type. This is in contrast to type I, inwhich P53 mutations are relatively uncommon andare largely confined to grade 3 tumors. The exactmechanism for the cause of this mutation is still un-clear. After DNA damage, nuclear P53 accumulatesand causes cell cycle arrest by inhibiting cyclin-D1phosphorylation of the Rb gene and therefore pro-moting apoptosis. Thus, mutated P53 results in a non-functional protein that accumulates in the cell andacts as a double negative inhibitor of the wild-typeP53, leading to propagation of aberrant cells [18].A much lower P53 mutational rate in type I tu-mors makes P53 a useful molecular marker that dis-tinguishes type I from type II endometrial cancers[19]. Overexpression of tumor P53 protein has beenstudied by immunohistochemistry; its prevalence inendometrial cancer reportedly ranged from 30% to80%. Several studies have concluded that the exami-nation of tumor P53 provides independent prognos-tic information [14, 15], while others have failed toshow such a relationship [7, 16, 17, 20].In our study, we found that 54.5% of type II tu-mors and 23.7% of type I tumors expressed P53 andthis difference was statistically significant. In previ-ous publications, the difference in P53 expressionbetween these two types of tumors has proved to bemore distinct [5, 14, 17, 21]. We noted an increasedP53 immunoexpression in type II tumors but P53immunoexpression was not found to be statisticallydifferent when surgical staging was analyzed. Studieshave correlated P53 immunoexpression with a de-crease in overall survival in patients with endometri-al tumors [22, 23]. In our study, P53 positivity wasalso associated with the risk of death.BCL-2 immunoexpression was observed in 57.9%in type I tumors and 36.4% of type II that we ana-lyzed. The literature describes rates of 30–85%[9, 16, 24], depending on the selection of patients andtype of lesion. Our study demonstrated a correlationbetween BCL-2 immunoexpression and tumor type,stage and patient survival. Several studies have shownincreased BCL-2 immunoexpression to be associat-ed with some favorable prognostic factors in type Itumors in initial stages [9, 10, 24, 25]. In our study,a statistically significant correlation between BCL-2and disease free survival was found in type I tumors.In conclusion, P53 immunoexpression was signif-icantly more frequent in type II tumors and correlat-Figure 4. Kaplan–Meier survival analysis concerningBCL-2 immunoexpression and cumulative survival in typeII endometrial cancer635Prognostic significance of P53 and BCL-2 in EC©Polish Society for Histochemistry and CytochemistryFolia Histochem Cytobiol. 201110.5603/FHC.2011.0086www.fhc.viamedica.pled with a decrease in overall survival in both types ofendometrial cancer. A positive correlation was ob-served between decreased BCL-2 immunoexpressionand patient survival in type I tumors. Thus, the prog-nostic value of these biomarkers in endometrial can-cer needs to be further investigated.References1. Reports based on data of National Cancer Registry. TheMaria Sklodowska-Curie Memorial Cancer Center, Depart-ment of Epidemiology and Cancer Prevention, National Can-cer Registry 2004, http://epid.coi.waw.pl/krn.2. Amant F, Moerman P, Neven P et al. Endometrial cancer.Lancet. 2005;366:491–505.3. Dobrzycka B, Terlikowski SJ, Mazurek A et al. Circulatingfree DNA, p53 antibody and mutations of KRAS gene inendometrial cancer. Int J Cancer. 2010;127:612–621.4. Dobrzycka B, Terlikowski SJ, Mazurek A et al. Mutations ofthe KRAS oncogene in endometrial hyperplasia and carci-noma. Folia Histochem Cytobiol. 2009;47:65–68.5. Engelsen IB, Akslen LA, Salvesen HB. Biologic markers inendometrial cancer treatment. APMIS. 2009;117:693–707.6. Ioachin E. Immunohistochemical tumour markers in endome-trial carcinoma. Eur J Gynaecol Oncol. 2005;26:363–371.7. Terlikowski S, Lenczewski A, Famulski W et al. Patterns ofimmunohistochemical staining for p53 expression in hyper-plastic endometrium and adenocarcinoma. Folia HistochemCytobiol. 2001;39(Suppl 2):195–196.8. Terlikowski S, Lenczewski A, Famulski W et al. Proliferativeactivity in endometrial hyperplasia and adenocarcinoma. Fo-lia Histochem Cytobiol. 2001;39:163–164.9. Porichi O, Nikolaidou ME, Apostolaki A et al. BCL-2, BAXand P53 expression profiles in endometrial carcinoma as stud-ied by real-time PCR and immunohistochemistry. Antican-cer Res. 2009;29:3977–3982.10. Appel ML, Edelweiss MI, Fleck J et al. P53 and BCL-2 asprognostic markers in endometrial carcinoma. Pathol OncolRes. 2008;14:23–30.11. Burton JL, Wells M. Recent advances in the histopathologyand molecular pathology of carcinoma of the endometrium.Histopathology. 1998;33:297–303.12. Lax SF. Molecular genetic changes in epithelial, stromal andmixed neoplasms of the endometrium. Pathology.2007;39:46–54.13. Abal M, Planaguma J, Gil-Moreno A et al. Molecular pa-thology of endometrial carcinoma: transcriptional signaturein endometrioid tumors. 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The frequency of p53,K-ras mutations, and microsatellite instability differs in uter-ine endometrioid and serous carcinoma: evidence of distinctmolecular genetic pathways. Cancer. 2000;88:814–824.20. Alkushi A, Lim P, Coldman A et al. Interpretation of p53immunoreactivity in endometrial carcinoma: establishinga clinically relevant cut-off level. Int J Gynecol Pathol.2004;23:129–137.21. Geisler JP, Wiemann MC, Zhou Z et al. p53 as a prognosticindicator in endometrial cancer. Gynecol Oncol. 1996;61:245–248.22. Jongen VH, Briët JM, de Jong RA et al. Aromatase, cycloox-ygenase 2, HER-2/neu, and p53 as prognostic factors in en-dometrioid endometrial cancer. Int J Gynecol Cancer.2009;19:670–676.23. Ito K, Watanabe K, Nasim S et al. Prognostic significance ofp53 overexpression in endometrial cancer. Cancer Res.1994;54:4667–4670.24. Kalogiannidis I, Bobos M, Papanikolaou A et al. Immuno-histochemical bcl-2 expression, p53 overexpression, PR andER status in endometrial carcinoma and survival outcomes.Eur J Gynaecol Oncol. 2008;29:19–25.25. Coppola D, Fu L, Nicosia SV et al. Prognostic significanceof p53, bcl-2, vimentin, and S100 protein-positive Langer-hans cells in endometrial carcinoma. Hum Pathol. 1998;29:455–462.Submitted: 18 August, 2010Accepted after reviews: 18 September, 2011

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The prognostic significance of the immunohistochemical expression of P53 and BCL-2 in endometrial cancer

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The prognostic significance of the immunohistochemical expression of P53 and BCL-2 in endometrial cancer

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