OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2016.4890 OL-0-0-4890 Articles Augmented expression of Polo-like kinase 1 is a strong predictor of shorter cancer-specific overall survival in early stage breast cancer at 15-year follow-up DonizyPiotr 1 HalonAgnieszka 1 SurowiakPawel 2 KaczorowskiMaciej 1 KozyraCyprian 3 MatkowskiRafal 4 5 Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw 50-556, Poland Department of Histology and Embryology, Wroclaw Medical University, Wroclaw 50-556, Poland Department of Statistics, Wroclaw University of Economics, Wroclaw 53-345, Poland Department of Oncology and Surgical Oncology, Wroclaw Medical University, Wroclaw 50-556, Poland Lower Silesian Oncology Centre, Breast Unit, Wroclaw 53-413, Poland Correspondence to: Mr. Piotr Donizy, Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Borowska 213, Wroclaw 50-556, Poland, E-mail: piotrdonizy@wp.pl 09 2016 20 07 2016 12 3 1667 1674 22052015 15042016 Copyright: © Donizy et al. 2016 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Polo-like kinase 1 (PLK1) is a serine-threonine kinase that plays a crucial role in the regulation of cell division. In addition, it acts as a modulator of the DNA damage response and as a novel factor in the maintenance of genome stability during DNA replication. The present study aimed to reveal the associations between PLK1 expression and clinicopathological features of patients with breast cancer (BC), particularly patient survival at 5-, 10- and 15-year follow-up. PLK1 expression was evaluated immunohistochemically in routine diagnostic tissue specimens from 83 patients treated radically for stage II BC. Kaplan-Meier analysis revealed a correlation between PLK1 overexpression and long-term survival. High PLK1 immunoreactivity was associated with shorter cancer-specific overall survival (CSOS) and disease-free survival (P=0.00001 and 0.00013, respectively). Multivariate analysis confirmed the negative prognostic significance of PLK1 overexpression for CSOS in all 83 patients (P=0.00030). Furthermore, analogous correlations were observed in both subgroups with and without nodal metastases (P=0.01400 and 0.01200, respectively). The present results indicate that PLK1 expression has a prognostic role in early BC. Immunohistochemical assessment of PLK1 reactivity may potentially become a qualifier for inclusion of PLK1 inhibitor therapy.

 Polo-like kinase 1 breast cancer prognosis survival analysis Introduction

Breast cancer (BC) is the second most common malignancy in the world, with estimated 1.67 million newly diagnosed cases and 522,000 associated mortalities worldwide in 2012 (http://globocan.iarc.fr; accessed May 1, 2015). Despite recent efforts to improve the detection rates and treatment of BC, the current situation, as reflected by disease statistics, is not favorable. A better understanding of the tumor's pathobiology will undoubtedly bring novel possibilities for treatment and diagnosis. In an attempt to offer the best medical approach possible for every single patient, modern medicine is evolving towards personalized therapies, which are characterized by a proper balance between the most radical approach possible while avoiding undesired side effects resulting from aggressive treatment. This attitude requires novel ideas regarding drug development and accurate stratification of the patients; therefore, novel prognostic factors are necessary.

Dysregulated cellular division is a key event in cancer initiation and progression. Polo-like kinases (PLKs) are a family of proteins that regulate the cell cycle (1). There are four PLKs with serine-threonine kinase activity in humans (PLK1-PLK4), whereas in PLK5 [which was first described in mice (2)] the kinase domain is truncated and does not possess any catalytic activity (3). However, PLK5 appears to participate in neuronal differentiation and act as a tumor suppressor in brain cancer (3). In addition, PLK1-PLK4, and particularly PLK2, display other roles beyond mitosis regulation (4,5).

PLK1, the best characterized protein of the PLK family, is a serine-threonine kinase that plays a crucial role in the regulation of cell division, centrosome maturation and duplication, assembly of the bipolar spindle, sister chromatid splitting, activation of the anaphase-promoting complex (APC), regulation of mitotic exit and induction of cytokinesis (1,6–14). PLK1 protein comprises two main domains: i) A conserved serine-threonine kinase domain at the N-terminus that is crucial for its kinase activity, and ii) a polo-box domain, a non-catalytic domain that is critical for its spatial distribution in the cells and its molecular interactions with specific substrates (12,13). PLK1 directly phosphorylates cell division cycle 27 protein (a component of the APC) and cyclin B1 (15,16), and together with other important signaling proteins such as p34 kinase, is responsible for mitotic progression (12).

PLK1 acts as a modulator of the DNA damage response and as a novel factor in the maintenance of genome stability during DNA replication (13). In response to DNA damage, the checkpoint kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) become activated and inhibit entry into mitosis via deactivation of cyclin-dependent kinase 1 (CDK1), which is the crucial kinase that promotes cell division (17). Effective resumption of cell cycle progression (arrested at G2/M) and mitotic entry upon successful repair of DNA damage are based on the activation of PLK1 by Aurora A/Bora-mediated phosphorylation (18,19). Additionally, activated PLK1 is involved in the enzymatic inactivation of WEE1 (a protein kinase that inhibits CDK1) (20) and elimination of claspin, which functions as a key adaptor protein for checkpoint kinase 1 activity (21). Cytophysiological downregulation of WEE1 and claspin by enhanced activity of PLK1 promotes CDK1 activation and leads to mitotic entry (20,21).

High PLK1 expression is observed within intensively proliferating normal tissues such as placenta and colonic epithelium (22), and in various types of cancer, including gastric (23), colorectal (24), hepatocellular (25), prostate (26), breast (27,28), ovarian (29) and non-small cell lung carcinomas (30). Notably, due to its position as a central controller of mitosis, PLK1 has become a potentially valuable target for antiproliferative therapies (31). Emerging experimental results are encouraging, and several anti-PLK1 agents are currently being investigated in clinical trials (32). No predictive factor has been identified thus far that could be used as a reliable qualifier to the potential inclusion of PLK1 inhibitor therapy in BC treatment. Similarly to the case of human epidermal growth factor receptor-2 (HER-2), immunohistochemical analysis of PLK1 expression in BC cells and the subsequent decision of initiating therapy or excluding the patient from therapy may be an alternative. This hypothesis requires verification in extensive, multicentre research studies.

Although great progress has been achieved since the initial characterization of human PLK1 >20 years ago (22,33), there is disagreement among researchers regarding the precise role of this kinase in cancer pathogenesis, and the prognostic significance of its expression in breast tumors has not been clearly established to date.

The present study reports an association between PLK1 expression and patient survival in 5-, 10- and 15-year follow-ups. In addition, an analysis of the correlations between PLK1 expression and other clinicopathological and histopathological features is provided.

 Materials and methods <sec> <title>Patients

Tissue samples were acquired from 83 radically treated patients with stage II ductal BC diagnosed between 1993 and 1994 in the Lower Silesian Oncology Centre (Wroclaw, Poland). The patients' mean age was 55.2 years. The study population was selected based on the availability of tissues. All patients underwent surgery (Madden mastectomy) with or without adjuvant treatment [27% of patients were treated by chemotherapy based on the CMF scheme (100 mg/m2 cyclophosphamide per day, days 1–14; 40 mg/m2 intravenous methotrexate, days 1 and 8; 500 mg/m2 intravenous fluorouracil, days 1 and 8; for 6 cyles of 28 days), which is no longer in use]. Following treatment, the patients were under continuous monitoring in the Lower Silesian Oncology Centre. Data regarding relapse and mortality were collected using medical documentation available in the Lower Silesian Oncology Centre. Overall survival (OS), cancer-specific overall survival (CSOS) and disease-free survival (DFS) rates were established for all patients. Table I contains detailed characteristics of the cohort. The present study was approved by the Institutional Review Board of Wroclaw Medical University.

 Tumor samples

Tumor specimens were fixed in 10% buffered formalin and embedded in paraffin. All hematoxylin and eosin stained sections were evaluated by two pathologists (P.D. and A.H., Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw). Tumor stages were assessed according to the tumor-node-metastasis classification system (34). Tumor grades were estimated according to the Scarff-Bloom-Richardson protocol (35), with the Elston-Ellis (36) modification (Table I).

 Immunohistochemistry

Immunohistochemical analyses were performed retrospectively on tissue samples collected for routine diagnostic purposes. Formalin-fixed, paraffin-embedded tissue sections were freshly prepared (4 µm thickness; Accu-Cut SRMTM; Sakura, Alphen aan den Rijn, The Netherlands). Immunohistochemistry was performed as previously described (37). For the detection of PLK1, a monoclonal mouse antibody against PLK1 (BD Transduction Laboratories™; BD Biosciences, Franklin Lakes, NJ, USA) was diluted 1:500 in the Antibody Diluent with Background Reducing Components (DakoCytomation; Dako, Glostrup, Denmark). For detection of estrogen receptor (ER), an optimally pre-diluted monoclonal mouse antibody was used (clone 1D5; DakoCytomation; Dako), while for detection of progesterone receptor (PgR), an optimally pre-diluted monoclonal antibody (clone PgR636; DakoCytomation; Dako) was used. For HER-2 detection, a semi-quantitative diagnostic immunohistochemical test was used (HercepTest™Kit; K5207; DakoCytomation; Dako). Tissue sections were incubated with the above antibodies for 1 h at room temperature. Subsequent incubations involved biotinylated antibodies (15 min, room temperature) and a streptavidin-biotinylated peroxidase complex (15 min, room temperature) (LSAB 2 System-HRP; DakoCytomation; Dako). As a chromogen, 3,3′-diaminobenzidine (DakoCytomation; Dako) was used (10 min, room temperature). All sections were counterstained with Mayer's hematoxylin. In each case, control reactions were included, in which the specific antibody was substituted by a primary mouse antibody (DakoCytomation; Dako), which served as a negative control.

 Evaluation of immunohistochemical reaction intensity

The intensity of the immunohistochemical reaction was assessed independently by two pathologists. In doubtful cases, a re-evaluation was performed using a double-headed microscope, (BX45; Olympus, Tokyo, Japan) and staining was discussed until a consensus was achieved.

PLK1 expression was evaluated using the semi-quantitative scale of the immunoreactive score (IRS), according to Remmele and Stegner with certain modifications (37,38), which considers the percentage of reactive cells (no staining=0, <25%=1, 25–50%=2, 51–75%=3 and >75%=4) and the intensity of staining (no staining=0, weak=1, intermediate=2 and strong=3), with the final result being the product of both variables. Consequently, nine possible scores (0, 1, 2, 3, 4, 6, 8, 9 and 12) were obtained.

PLK1 expression was only observed in tumor tissues of BC specimens with cytoplasmic localization. Normal breast tissues were characterized by no or weak cytoplasmic PLK1 immunoreactivity.

For subsequent statistical analyses, a two-grade scale system was applied, allocating 0 points for expression of PLK1 <8 (low PLK1 immunoreactivity) and 1 for expression of PLK1 ≥8 (high PLK1 immunoreactivity). Definition of these two groups and determination of the cut-off point is a specific consensus of histopathological observations and statistical analyses.

Evaluation of ER, PgR and HER-2 expression was performed using standard methods described in a previous study (36).

 Statistical analysis

Statistical analysis was performed using the Statistica 10.0 software package (StatSoft Inc., Tulsa, OK, USA). OS was defined as the time between primary surgical treatment and mortality, and it was censored at the last follow-up for those patients who were alive. DFS was defined as the time between primary surgical treatment and date of relapse or mortality, whichever occurred first. DFS was censored at the last follow-up for patients who survived without disease recurrence. CSOS was defined as the time between primary surgical treatment and cancer-associated mortality, and was censored at the last follow-up for surviving patients.

To analyze the associations between PLK1 protein expression and clinicopathological parameters, the Pearson linear correlation coefficient in case of quantitative variables, the Kendall rank correlation in case of ordinal variables, the Pearson χ2 test of independence in case of categorical variables and the exact Fisher test in case of 2×2 tables, were used. Differences between two groups were tested with the Mann-Whitney U test, while the log-rank test was used for comparison of survival in two groups. The OS rate was estimated by the Kaplan-Meier method, and the influence of explanatory variables on mortality risk was analyzed by Cox proportional hazard regression and logistic regression in case of binary survival. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>PLK1 immunostaining in BC specimens

PLK1 expression defined as IRS >0 was detected in all 83 BC patients. The average IRS was 6.55±3.10, and the median was 6.00. For statistical analysis, augmented immunoreactivity of PLK1 was defined as IRS ≥8 (38 patients, 45.8%), while low immunoreactivity was assigned to IRS=0–6 (45 patients, 54.2%) (Fig. 1).

 Association between PLK1 expression and clinicopathological parameters

Overexpression of PLK1 and high intensity of immunohistochemical reaction were significantly correlated with the presence of regional lymph node metastases (P=0.03700 and 0.02000, respectively) (Table I). Disease recurrence was observed more frequently in patients with increased PLK1 expression and with high intensity of PLK1 immunoreactivity (P<0.00100 and P=0.00100, respectively). Paradoxically, increased PLK1 expression and high percentage of PLK1+ cells were associated with lower histological grade (P=0.01400 and 0.00100, respectively). No significant correlations were observed between PLK1 expression and hormone receptor/HER-2 status, primary tumor size, menopausal status or age at the time of diagnosis (Table I).

 PLK1 immunoreactivity and patient survival at 5-, 10- and 15-year follow-ups

Univariate logistic regression analysis of PLK1 expression in the context of 5-, 10- and 15-year survival revealed highly negative prognostic significance of PLK1 overexpression in patients with early stage BC in all the follow-up periods analyzed (Table II).

Kaplan-Meier analysis confirmed the correlation of PLK1 overexpression with long-term survival, as high PLK1 immunoreactivity (IRS ≥8) was associated with shorter CSOS and DFS (P=0.00001 and 0.00013, respectively) (Fig. 2A and B). Additionally, high PLK1 immunoreactivity was correlated with shorter CSOS and DFS in patients without local lymph node metastases (P=0.00110 and 0.00900, respectively) (Fig. 2C and D) and in patients with diagnosed nodal metastatic foci (P=0.00900 and 0.03000, respectively) (Fig. 2E and F).

 Multivariate Cox regression analysis

In the multivariate Cox regression analysis, two clinicopathological parameters were noticed to have independent prognostic value in patients with early stage BC, namely high expression of PLK1 (P=0.00030) and presence of local lymph node metastases (P=0.00300). Other clinicopathological features had no significance in the multivariate Cox model.

Since lymph node metastases had a significant prognostic impact, multivariate analysis was performed individually in N− and N+ patients (Table II). It was demonstrated that, in both lymph node-negative and positive groups, high expression of PLK1 was an independent unfavorable prognostic factor (P=0.01200 and 0.01400, respectively), which confirms the findings of univariate analysis.

 Discussion

In the present study, a homogeneous group of patients with stage II invasive ductal BC was investigated with regard to expression levels of PLK1 and patient survival in a 15-year follow-up period. The associations between PLK1 reactivity in BC specimens and the status of HER-2 and steroid receptors were also evaluated.

Overexpression of PLK1 (defined as IRS ≥8) was detected in 45.8% of patients (38 patients), while low immunoreactivity of PLK1 was observed in 54.2% of patients (45 patients). PLK1 expression was only observed in the tumoral compartment of BC specimens, with cytoplasmic localization. With regard to the cytoplasmic expression pattern of PLK1, the present results were similar to those reported by other studies (27,28). By contrast, the findings regarding the cut-off value for high PLK1 immunoreactivity and the incidence of PLK1 overexpression were less concordant (27,28). King et al (28) demonstrated PLK1 overexpression in only 11% of analyzed patients, whereas Weichert et al (27) reported overexpression in 42.2% cases of BC, a value close to the present observations (45.8%). Likely reasons for these dissimilarities are methodological differences in PLK1 expression assessment between the studies and a highly homogenous study population (stage II, according to the Union for International Cancer Control classification) in the present study (34).

In the current study, overexpression of PLK1 was significantly correlated with the presence of regional lymph node metastases (P=0.03700) and disease recurrence (P<0.00100). Kaplan-Meier analysis confirmed the correlation of PLK1 overexpression with long-term survival, as high PLK1 immunoreactivity was strongly associated with shorter CSOS and DFS (P=0.00001 and 0.00013, respectively). In a multivariate Cox regression analysis, two clinicopathological parameters were observed to have independent prognostic value in patients with early stage BC: High expression of PLK1 (P=0.00030) and presence of regional lymph node metastases (P=0.00300).

Highly negative impact of increased PLK1 expression on patient prognosis was also observed by King et al (28), who demonstrated significantly shorter OS of patients with PLK1 overexpression in their analysis of 215 subjects. In addition, a positive correlation between PLK1 expression and the presence of a mutant version of the tumor protein p53 gene was also revealed in that study (28). Weichert et al (27) did not confirm the prognostic significance of enhanced PLK1 immunoreactivity in BC cells, and only PLK3 overexpression was observed by the authors to be a negative predictor of OS and recurrence-free survival.

An important point in the interpretation of the present results is the significant correlation between PLK1 overexpression and the presence of regional lymph node metastases, which is commonly accepted as an independent predictor of negative prognosis (38). King et al (28) and Weichert et al (27) did not observe any significant associations between increased PLK1 immunoreactivity and regional nodal metastases. The absence of associations between PLK1 overexpression and PgR/HER-2 status in the current results are in agreement with those of other authors (27,28). Notably, King et al (28) and Weichert et al (27) demonstrated that negative ER status and high histological grade correlated with PLK1 overexpression, which was not confirmed in the present study. This is probably due to the highly homogenous population (comprising only early BC patients) in the current study, whereas the study groups in the aforementioned reports contained patients in all stages of the disease.

Another aspect worth considering is the role of PLK1 expression as a potential marker of cell proliferation, since strong expression of PLK1 (which has been associated with enhanced mitotic activity) is detectable in actively proliferating cells (those in phase G2/M) (39). In the present study and in the study conducted by Weichert et al (27), there were cases of BC in which 100% of cells exhibited strong PLK1 immunoreactivity. This observation is difficult to interpret and requires further investigation. PLK1 overexpression is closely associated with the G2/M phase of the cell cycle in in vitro models (40). However, such a remarkably high proportion of PLK1+ cells does not necessarily imply that all the positive cells are in the G2/M phase (which is the active phase of proliferation) at the same time. The above observation may indicate a pleiotropic significance of PLK1 in cytophysiology; thus, its expression may not only be a symptom of ongoing cellular divisions, but may also reflect a cellular response to DNA damage in cancer cells and the subsequent attempts to repair it by numerous enzymes, including ATM, ATR and poly(ADP-ribose) polymerase 1 (PARP-1). This postulate is in line with the results of the present study, which identified a positive correlation between enhanced PLK1 and PARP-1 expression in BC cells (data not shown).

Additionally, PLK1 overexpression in cancer cells may result from chromosomal overrepresentation of the PLK1 gene locus, which leads to increased protein production. This is consistent with the observations of Tirkkonen et al (41), who detected chromosomal amplification of the 16p12 region (which contains the PLK1 locus) in 38% of BC patients, a rate that is close to the 45.8% of tumors overexpressing PLK1 detected in the present study.

In conclusion, there is a significant and independent association between PLK1 overexpression and unfavorable prognosis in the 15-year follow-up of early BC patients. The results of the present study suggest a potential role for PLK1 in the progression of BC. The present findings may aid to generate molecular targeted therapies based on PLK1 inhibitors.

 Acknowledgements

The present study was supported by funding from the Wroclaw Medical University (Wroclaw, Poland; research grant nos. ST-593 and Pbmn157).

 References GloverDMHaganIMTavaresAAPolo-like kinases: A team that plays throughout mitosisGenes Dev1237773787199810.1101/gad.12.24.37779869630 AndrysikZBernsteinWZDengLMyerDLLiYQTischfieldJAStambrookPJel BahassiMThe novel mouse polo-like kinase 5 responds to DNA damage and localizes in the nucleolusNucleic Acids Res3829312943201010.1093/nar/gkq01120100802 de CárcerGEscobarBHigueroAMGarcíaLAnsónAPérezGMollejoMManningGMeléndezBAbad-RodríguezJMalumbresMPlk5, a polo box domain-only protein with specific roles in neuron differentiation and glioblastoma suppressionMol Cell Biol3112251239201110.1128/MCB.00607-1021245385 de CárcerGManningGMalumbresMFrom Plk1 to Plk5: Functional evolution of polo-like kinasesCell Cycle1022552262201110.4161/cc.10.14.1649421654194 SeeburgDPPakDShengMPolo-like kinases in the nervous systemOncogene24292298200510.1038/sj.onc.120827715640845 HansenDVLoktevAVBanKHJacksonPKPlk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1Mol Biol Cell1556235634200410.1091/mbc.E04-07-059815469984 GolsteynRMMundtKEFryAMNiggEACell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle functionJ Cell Biol12916171628199510.1083/jcb.129.6.16177790358 LaneHANiggEAAntibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomesJ Cell Biol13517011713199610.1083/jcb.135.6.17018991084 PetronczkiMGlotzerMKrautNPetersJMPolo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindleDev Cell12713725200710.1016/j.devcel.2007.03.01317488623 LoweryDMLimDYaffeMBStructure and function of Polo-like kinasesOncogene24248259200510.1038/sj.onc.120828015640840 van VugtMAMedemaRHGetting in and out of mitosis with Polo-like kinase-1Oncogene2428442859200510.1038/sj.onc.120861715838519 TakaiNHamanakaRYoshimatsuJMiyakawaIPolo-like kinases (Plks) and cancerOncogene24287291200510.1038/sj.onc.120827215640844 TakakiTTrenzKCostanzoVPetronczkiMPolo-like kinase 1 reaches beyond mitosis-cytokinesis, DNA damage response, and developmentCurr Opin Cell Biol20650660200810.1016/j.ceb.2008.10.00519000759 el BahassiMPolo-like kinases and DNA damage checkpoint: Beyond the traditional mitotic functionsExp Biol Med (Maywood)236648657201110.1258/ebm.2011.01101121558091 KotaniSTugendreichSFujiiMJorgensenPMWatanabeNHoogCHieterPTodokoroKPKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progressionMol Cell1371380199810.1016/S1097-2765(00)80037-49660921 NiggEAMitotic kinases as regulators of cell division and its checkpointsNat Rev Mol Cell Biol22132200110.1038/3504809611413462 BartekJLukasJDNA damage checkpoints: From initiation to recovery or adaptationCurr Opin Cell Biol19238245200710.1016/j.ceb.2007.02.00917303408 MacůrekLLindqvistALimDLampsonMAKlompmakerRFreireRClouinCTaylorSSYaffeMBMedemaRHPolo-like kinase-1 is activated by aurora A to promote checkpoint recoveryNature455119123200810.1038/nature0718518615013 SekiACoppingerJAJangCYYatesJRFangGBora and the kinase Aurora A cooperatively activate the kinase Plk1 and control mitotic entryScience32016551658200810.1126/science.115742518566290 van VugtMABrásAMedemaRHPolo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cellsMol Cell15799811200410.1016/j.molcel.2004.07.01515350223 MamelyIvan VugtMASmitsVASempleJILemmensBPerrakisAMedemaRHFreireRPolo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recoveryCurr Biol1619501955200610.1016/j.cub.2006.08.02616934469 HoltrichUWolfGBräuningerAKarnTBöhmeBRübsamen-WaigmannHStrebhardtKInduction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumorsProc Natl Acad Sci USA9117361740199410.1073/pnas.91.5.17368127874 JangYJKimYSKimWHOncogenic effect of Polo-like kinase 1 expression in human gastric carcinomasInt J Oncol29589594200616865274 TakahashiTSanoBNagataTKatoHSugiyamaYKuniedaKKimuraMOkanoYSajiSPolo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancersCancer Sci94148152200310.1111/j.1349-7006.2003.tb01411.x12708489 HeZLZhengHLinHMiaoXYZhongDWOverexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patientsWorld J Gastroenterol1541774182200910.3748/wjg.15.417719725153 WeichertWSchmidtMGekelerVDenkertCStephanCJungKLoeningSDietelMKristiansenGPolo-like kinase 1 is overexpressed in prostate cancer and linked to higher tumor gradesProstate60240245200410.1002/pros.2005015176053 WeichertWKristiansenGWinzerKJSchmidtMGekelerVNoskeAMüllerBMNiesporekSDietelMDenkertCPolo-like kinase isoforms in breast cancer: Expression patterns and prognostic implicationsVirchows Arch446442450200510.1007/s00428-005-1212-815785925 KingSIPurdieCABraySEQuinlanPRJordanLBThompsonAMMeekDWImmunohistochemical detection of Polo-like kinase-1 (PLK1) in primary breast cancer is associated with TP53 mutation and poor clinical outcomeBreast Cancer Res14R40201210.1186/bcr313622405092 WeichertWDenkertCSchmidtMGekelerVWolfGKöbelMDietelMHauptmannSPolo-like kinase isoform expression is a prognostic factor in ovarian carcinomaBr J Cancer90815821200410.1038/sj.bjc.660161014970859 WangZXXueDLiuZLLuBBBianHBPanXYinYMOverexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancerInt J Biochem Cell Biol44200210201210.1016/j.biocel.2011.10.01722064247 WeißLEfferthTPolo-like kinase 1 as target for cancer therapyExp Hematol Oncol138201210.1186/2162-3619-1-3823227884 YimHCurrent clinical trials with polo-like kinase 1 inhibitors in solid tumorsAnticancer Drugs249991006201310.1097/CAD.000000000000000723949254 HamanakaRMaloidSSmithMRO'ConnellCDLongoDLFerrisDKCloning and characterization of human and murine homologues of the Drosophila polo serine-threonine kinaseCell Growth Differ524925719948018557 BloomHJRichardsonWWHistological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 yearsBr J Cancer11359377195710.1038/bjc.1957.4313499785 SobinLHGospodarowiczMKWittekindCTNM Classification of Malignant Tumours7thWiley-BlackwellHoboken, NJ2009 ElstonCWEllisIOPathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: Experience from a large study with long-term follow-upHistopathology19403410199110.1111/j.1365-2559.1991.tb00229.x1757079 HalonADonizyPSurowiakPMatkowskiRERM/Rho protein expression in ductal breast cancer: A 15 year follow-upCell Oncol (Dordr)36181190201310.1007/s13402-013-0125-923420497 FitzgibbonsPL1PageDLWeaverDThorADAllredDCClarkGMRubySGO'MalleyFSimpsonJFConnollyJLPrognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999Arch Pathol Lab Med124966978200010888772 DegenhardtYLampkinTTargeting Polo-like kinase in cancer therapyClin Cancer Res16384389201010.1158/1078-0432.CCR-09-138020068088 RemmeleWStegnerHERecommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissuePathologe81381401987(In German)3303008 TirkkonenMTannerMKarhuRKallioniemiAIsolaJKallioniemiOPMolecular cytogenetics of primary breast cancer by CGHGenes Chromosomes Cancer21177184199810.1002/(SICI)1098-2264(199803)21:3<177::AID-GCC1>3.3.CO;2-C9523192

Immunohistochemical analysis of PLK1 expression in BC cells. (A) Lack of PLK-1 expression in BC cells (IRS=0; magnification, ×400; hematoxylin staining). (B) Intermediate level of cytoplasmic PLK1 expression in BC cells (IRS=6; magnification, ×200; hematoxylin staining). (C and D) High expression of PLK1 in BC cells of two different tumors (IRS=12; magnification, ×600; hematoxylin staining). PLK1, polo-like kinase 1; BC, breast cancer; IRS, immunoreactive score.

PLK1 immunoreactivity and patient survival. High PLK1 immunoreactivity (IRS ≥8) was associated with shorter (A) CSOS (P=0.00001) and (B) DFS (P=0.00013). High PLK1 immunoreactivity was associated with shorter (C) CSOS (P=0.00110) and (D) DFS (P=0.00900) in patients without regional lymph node metastases. High PLK1 immunoreactivity was associated with shorter (E) CSOS (P=0.00900) and (F) DFS (P=0.03000) in patients with diagnosed nodal metastatic foci. PLK1, polo-like kinase 1; N, lymph nose metastasis; gr., group; CSOS, cancer-specific overall survival; DFS, disease-free survival.

Patient and tumor characteristics, and their association with PLK1 immunoreactivity in breast cancer patients.

Parameters of PLK1 immunoreactivity
Patient characteristics No. (%) % Intensity IRS High expression of PLK1 (IRS ≥8)
All patients 83 (100.0) 0.888 0.204 0.480b 0.982c
Age (years)a
  Mean, 55.2±10.3; median: 55
  Median, 55
Menopausec
  Premenopausal 27 (32.5) 0.316 0.392 0.935 0.296d
  Postmenopausal 56 (67.5)
TNM stage according to UICCc 0.244 0.674 0.879 0.570d
  II A 33 (39.8)
  II B 50 (60.2)
Tumor size (pT)a (mm) 0.540 0.585 0.969 0.280c
  Mean, 31.0±12.3
  Median, 30
Nodal metastases (N)c 0.232 0.020 0.006 0.037d
  N_ 47 (56.6)
  N+ 36 (43.4)
Gradingc 0.001 0.746 0.057 0.014d
  G2 59 (71.1)
  G3 24 (28.9)
ER statusa 0.182 0.561 0.363 0.464c
  Negative 22 (26.5)
  Positive 61 (73.5)
PgR statusa 0.169 0.374 0.894 0.831c
  Negative 22 (26.5)
  Positive 61 (73.5)
HER-2 statusa 0.735 0.714 0.875 0.646c
  Negative 64 (77.1)
  Positive 19 (22.9)
Recurrencec 0.394 0.001 <0.001 <0.001d
  Yes 32 (38.6)
  No 51 (61.4)

P-value of Kendall's tau rank correlation coefficient.

P-value of Pearson's correlation.

P-value of Mann-Whitney's U test.

P-value of Fisher's test. PLK1, polo-like kinase 1; IRS, immunoreactive score; ER, estrogen receptor; PgR, progesterone receptor; HER-2, human epidermal growth factor receptor-2; TNM, tumor-node-metastasis; UICCc, Union for International Cancer Control classification.

Univariate analysis of correlations between immunohistochemical parameters of PLK1 expression and 5-, 10- and 15-year CSOS, and multivariate Cox regression analysis of PLK1 expression and 15-year CSOS in groups with and without lymph node metastases and in the whole cohort of patients.

A, Univariate logistic regression
5-year survival 10-year survival 15-year survival
Parameters of PLK1 expression P-value OR (95% CI) P-value OR (95% CI) P-value OR (95% CI)
Positive cells (%) 0.20700 1.85 (0.71–4.87) 0.18800 1.62 (0.79–3.33) 0.11400 1.79 (0.87–3.68)
Intensity 0.01200 3.14 (1.29–7.65) 0.00200 3.33 (1.59–7.01) 0.00040 4.51 (2.01–10.10)
IRS 0.00700 1.35 (1.09–1.67) 0.00080 1.40 (1.16–1.70) 0.00020 1.54 (1.24–1.92)
High expression (IRS ≥8) 0.00500 9.92 (2.01–49.05) 0.00060 7.80 (2.48–24.51) 0.00010 12.18 (3.75–39.62)
B, Multivariate Cox regression analysis of 15-year survival
All patients Without lymph node metastases With lymph node metastases
Clinicopathological parameters P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI)
High expression of PLK1 0.00030 6.13 (2.30–16.33) 0.01200 19.21 (1.91–193.28) 0.01400 4.02 (1.32–12.20)
Tumor size (pT) 0.12100 1.03 (0.99–1.07) 0.05100 1.07 (1.00–1.14) 0.52600 1.01 (0.97–1.06)
Lymph node metastases 0.00300 3.57 (1.55–8.24) – – – –

CSOS, cancer-specific overall survival; PLK1, polo-like kinase 1; IRS, immunoreactive score; OR, odds ratio; CI, confidence interval; HR, hazard ratio.