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Gynecologic Oncology
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Histological and immunohistochemical profiles predict lymph nodestatus in women with low-intermediate risk endometrial cancer
Marcos Ballester a,b,c,⁎, Geoffroy Canlorbe a, Annie Cortez d, Julie Gonin d, Enora Laas a, Sofiane Bendifallah a,Olivier Graesslin e, Emile Daraï a,f
a Service de Gynécologie-Obstétrique, Hôpital Tenon, AP-HP, Paris, Franceb Institut Universitaire de Cancérologie, Université Pierre et Marie Curie, Paris 6, Francec ER2 UPMC, Université Pierre et Marie Curie, Paris 6, Franced Service d'Anatomie Pathologie, Hôpital Tenon, AP-HP, Paris, Francee Service de Gynécologie-Obstétrique, Hôpital Maison Blanche, CHU de Reims, Francef UMRS 938, Université Pierre et Marie Curie, Paris 6, France
H I G H L I G H T S
• Predictive models can be helpful to assess lymph node status in low-intermediate risk endometrial cancer.• Additional immunohistochemical markers coupled with histological criteria allowed a better prediction with a misclassification rate of 13%.• The positive predictive value of the recursive partitioning model was 92%.• This can help physicians to better adapt surgical staging and adjuvant therapies
⁎ Corresponding author at: Service de Gynécologie-ObsParis, France.
Objective. The aim of this study was to build a model to predict the risk of lymph node metastases(LNM) in women with low- or intermediate-risk endometrial cancer (EC) using histological and immuno-histochemical markers.
Methods. Samples were collected from 68 women with low- or intermediate-risk EC. European Societyof Medical Oncology (ESMO) risk group, lymphovascular space involvement (LVSI), immunostainingexpressions of Estrogen receptor (ER) and Progesteron receptor (PR) were used to build a recursivepartitioning model to predict final lymph node status.
Results. The number of women with final low- and intermediate risk EC was 34 (50%) each. LVSI waspresent in 7 women with low-risk (20%) and 28 (80%) with intermediate-risk EC. Nineteen women(28%) had LNM at final histology. A lower immunostaining of ER (p = 0.02) and PR (p = 0.03) wasfound in women with LNM compared with those without. Women were correctly classified by the
model in 87% of cases; among the 56 women without LNM that were predicted, 48 (86%) had no LNM atfinal histology. Among the 12 women with LNM predicted, 11 (92%) had LNM at final histology.
Conclusions. Our results show that lymph node status can be predicted with a relatively high accuracyin women with low- or intermediate-risk EC. This can help physicians to better adapt surgical staging andadjuvant therapies.
Endometrial cancer (EC) is the most common malignancy of thefemale reproductive tract and the fourth most common cancer overall[1]. Women with stage I disease, representing almost 75% of cases,have an overall survival of 95% [2]. For early stage EC, three risk
tétrique, Hôpital Tenon, AP-HP,
llester).
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groups with regard to disease relapse and survival have been defined:low, intermediate or high risk. However, there is currently no consen-sus on the most accurate criteria to define risk groups [2–4].
Lymph node status, itself related to histological type and gradeand depth of myometrial invasion, is a major prognostic factor of sur-vival of EC [2]. Previous studies have reported that 5% of women withlow-risk and 10% of women with intermediate-risk EC have lymphnode metastases justifying systematic assessment of lymph node sta-tus [5]. However, two randomized trials have recently shown thatpelvic lymphadenectomy has no impact on overall survival in early
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stage EC, whereas it increases postoperative morbidity [6,7]. There-fore, French guidelines have recently been modified and pelviclymphadenectomy is no longer recommended in women with low-or intermediate-risk EC [8]. In contrast, Ballester et al. found thatusing sentinel node biopsy, lymph node metastases were found in10% of women with low-risk and 15% of women with intermediate-risk EC [9]. However, decisions for nodal staging are taken preopera-tively based on endometrial biopsy and depth of myometrial invasionon Magnetic Resonance Imaging (MRI). In this specific setting, severalstudies have highlighted important discrepancies between preopera-tive and final histology [10]. Hence, additional tools are needed toselect women with low- or intermediate-risk EC with increased riskof lymph node metastases to adapt surgical staging and adjuvanttherapies.
The aim of this study was to evaluate whether histological and im-munohistochemical profiles of low-intermediate risk EC can predictlymph node status.
Materials and methods
Patients
This cohort study included 68 women with primary EC whounderwent surgical treatment with at least total hysterectomy withbilateral salpingo-oophorectomy and pelvic lymphadenectomy fromJune 2006 to December 2012 in the Department of Gynecology ofTenon Hospital (Paris). All the women had undergone a preoperativeendometrial biopsy and preoperative MRI to assess disease stage.Women were staged on the basis of final pathological findingsaccording to the 2009 International Federation of Gynecology andObstetrics (FIGO) classification [11].
Medical records were reviewed to determine age, surgical proce-dure, histological type and tumor grade, lymphovascular space inva-sion (LVSI) and depth of myometrial invasion on final histology.All the tissue samples were obtained with full and informed patientconsent. The research protocol was approved by the ConsultativeCommittee for Protection of Persons in Biomedical Research of Paris6 (France).
Endometrial cancer risk groups
As previously reported, histological type I corresponds toendometrioid tumors whatever the histological grade. Cases of his-tological type 2 including clear cell carcinomas, serous carcinomasand carcinosarcomas were excluded from the study [2].
Only women with final low- and intermediate-risk EC wereincluded in the present study. These two risk groups of EC weredefined as follows: low-risk (type 1 EC FIGO stage IA grade 1 or 2);intermediate-risk (type 1 EC, FIGO stage IA grade 3, or FIGO stage IBgrade 1 or 2) [2].
Grade 1 was defined by 5% or less nonsquamous, nonmorular solidgrowth pattern; grade 2 by 6% to 50% nonsquamous, nonmorulargrowth pattern; and grade 3 by more than 50% nonsquamous,nonmorular growth pattern [8]. Bizarre nuclear atypia should raisethe grade by one.
A tumor was considered LVSI-positive when tumor emboli werenoted within a space clearly lined by endothelial cells [12].
Immunohistochemistry
Tissues were fixed immediately in formalin (10%) and thenprocessed as paraffin blocks. Three micrometer-thick sections offormalin-fixed tissues were deparaffinated in a xylene substitute(EZ Prepw 1x ref 950102) and rehydrated through a graded seriesof ethanol solutions. Immunohistochemical staining was performedon paraffin sections with rabbit monoclonal antibodies directed
against estrogen receptors (ER) (prediluated, SP1, Ventana MedicalSystems, USA) and progesterone receptors (PR) (prediluated, 1E2,Ventana Medical Systems, USA). The sections were immunostainedusing the Ventana Benchmark XTw automated immunohistochemistrysystem (Ultra-ViewTM, Universal DAB-Ventana). An antigen retrievalprocedure was run (Dako Target Retrieval Solution; 98 °C, 20 min)prior to ER and PR staining. This automated procedure is based on an in-direct biotin-avidin system. A universal biotinylated immunoglobulinwas used as the secondary antibody, diaminobenzidine as the substrateand hematoxylin as the counterstain. Positive controls were sections ofhuman breast tissue for ER and PR [13].
Statistical analysis
For purposes of comparison, we separated the women into twoEuropean Society of Medical Oncology (ESMO) subgroups (low- vs.intermediate-risk EC).
For semi-quantitative analysis the percentage of stained epithelialcells was calculated by two independent observers. Each observerexamined the slides at least twice. Differences in opinion betweenthe observers were resolved by discussion microscope.
For qualitative analysis, we calculated optimal cut-offs for eachmarker to correlate semi-quantitative expression and final nodal sta-tus. Optimal cut-off for immunostaining level was determined by aminimal p-value approach. This involved dichotomizing the immuno-staining level into dummy variables with a cut-off every five units ofits range of values. Chi-square tests comparing the rate of womenwith or without lymph node metastases for every dummy variablewere then calculated. The cut-off with the minimal p-value was chosenas the optimal cut-off for this variable. Only samples with semi-quantitative expression (percentage of stained epithelial cells) ≥ optimalcut-off were considered positive.
We developed a recursive partitioning (RP) model to determineER, PR optimal cut-offs coupled with ESMO group and LVSI inpredicting final nodal status. RP is a technique that can be appliedto mine large data sets in order to uncover hidden patterns withinthe data and to reveal statistically significant subgroupings. At eachstep, the RP program optimally separates women into homogeneousgroups corresponding, in this case, to histological and immunohisto-chemical profiles. We internally validated our RP model using aBootstrap method which is based on a resampling obtained by draw-ing randomly with replacement from the original data set (1000resamplings were performed). To build the RP model, we analyzedlow- versus intermediate- risk EC.
Statistical analysis was based on Student's t-test and the Mann–Whitney test for parametric and nonparametric continuous variables,respectively, and the Chi-square test or Fisher's exact test, as appro-priate, for categorical variables. Values of p b 0.05 were consideredto denote significant differences.
Data were managed with an Excel database (Microsoft, Redmond,WA) and analyzed using R 2.15 software, available online.
Results
A total of 68 women were included in the study. Final histologicalgrade was 1, 2 and 3 in 32 cases (47 %) 29 cases (43 %) and 7 cases(10%), respectively. Depth of myometrial invasion was b50 % in 36cases (53 %) and ≥ 50 % in 32 cases (47 %). The number of womenwith final low- and intermediate risk EC was 34 (50%) each. LVSIwas present in 25 women (37 %), of whom 7 (20%) had a low-riskEC and 28 (80%) an intermediate-risk EC. A pelvic lymphadenectomywas performed in all women (37 cases (55%)) with associated senti-nel node biopsy. Nineteen women (28%) had metastatic lymph nodesat final histology; 3/34 (9%) in the low-risk group and 16/34 (47%)in the intermediate-risk group. Among the women with meta-static lymph nodes, macrometastases, micrometastases and isolated
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tumor cells (ITC) were found in 14 cases (74%), three cases (16%) andtwo cases (10%), respectively.
ER and PR immunostaining in low-intermediate risk EC
The results of semi-quantitative immunostaining of ER, PR aresummarized in Fig. 1. The semi-quantitative expression of eachmarker was significantly correlated with the final lymph node status.A lower immunostaining of ER (p = 0.02) and PR (p = 0.03) wasfound in women with lymph node metastases compared with thosewithout lymph node metastases.
Optimal cut-offs denoting the strongest correlation betweensemi-quantitative expression of ER and PR and final lymph nodestatus are summarized in Fig. 2. The positivity cut-offs defined were30% for ER and 15% for PR.
Using the cut-offs previously determined, we compared ER andPR expression between women with and without lymph node metas-tases: ER ≥ 30% immunostaining was more common in women with-out lymph node metastases (69%, p = 0.001) compared with thosewith lymph node metastases (37%); and PR ≥ 15% immunostainingmore common in women without lymph node metastases (73%,p = 0.03) compared with those with lymph node metastases (42%).Women with intermediate-risk EC were more likely to have lymphnode metastases compared with those with low-risk (p = 0.001).Women with LVSI were more likely to have lymph node metastasesat final histology compared with those without (p = 0.001) (Table 1).
Recursive partitioning model
An RP model based on the ESMO groups (low- vs. intermediate-risk EC), the presence of LVSI and previously determined cut-offs forER and PR was built (Fig. 3). The RP model predicted the absence oflymph node metastases in 100% of cases when ER was over expressed(above 30%) in low-risk EC. In contrast, the model found that theprobability of lymph node metastases was 88% in intermediate-riskEC when there was LVSI and low PR expression (below 15%).
In the training cohort, women were correctly classified in 87% ofcases; among the 56 women without lymph node metastases thatwere predicted by the RP model, 48 (86%) had no lymph nodesat final histology (i.e. negative predictive value). Among the 12women with lymph node metastases predicted by the RP model, 11(92%) had lymph node metastases at final histology (i.e. positivepredictive value). The sensitivity and specificity of the model were58% and 98%, respectively.
To internally validate the RP model, we used the bootstrapmethod with 1000 resamplings. The misclassification rate was 20%(95%CI: 19.1 to 20.6).
Fig. 1. Comparison of semi-quantitative estrogen and progesteron receptors im
Discussion
Our results show that in low- and intermediate-risk EC,womenwithlymph node metastases have different histological and immunohisto-chemical profiles compared with those without metastatic lymphnodes. Moreover, we found that final lymph node status can be accu-rately predicted using a combination of routine histological and immu-nohistochemical markers.
The main challenge for physicians faced with women withlow-intermediate risk EC is when to opt for lymphadenectomy. Torespond to this issue, we built a predictive model to assess lymphnode status using conventional histology coupled with immunohisto-chemistry. For women with low-risk EC, ER expression was ableto differentiate patients with low risk of metastatic lymph nodes.These results are in agreement with those of Yamauchi et al. whofound that lymph node metastases were more frequent in womenwith ER and PR negative EC [14]. Moreover, Markova et al. foundthat steroid receptor expression could contribute to the identificationof high risk groups [15]. In addition, we found that the combination ofPR with ER expression resulted in accurate prediction of lymph nodestatus in women with ER over expression. This is in accordance withprevious studies showing that PR immunostaining is an independentprognostic factor for survival [16,17]. Several studies have previouslyfocused on the interest of additional immunohistochemical markersto better characterize histological EC phenotypes. A relation hasbeen found between ER and PR immunostaining and good differenti-ation with better prognostic tumors [15,18–22]. However, in previousstudies, cut-offs determining positive immunostaining were chosenarbitrarily or according to previously published criteria. Hence,cut-offs for ER and PR positivity varied from 1% to 50% [15,19,23,24].In contrast, in our study, optimal cut-off for immunostaining levelwas determined by a minimal p-value approach allowing for greaterstrength and better reproducibility. Moreover, in a previous studyon immunohistochemical cut-off points for dividing breast cancersinto hormone receptor negatives and positives, Jalava et al. foundthat the optimal cut-off point may be different from one patientgroup to another [25]. This is in accordance with our results ascut-offs were determined to distinguish women with or withoutlymph nodes metastases among tumors from the same histologicalsubtype. Second, in most of the studies, immunohistochemical markerswere reported as independent factors. However, Clarke et al. previouslyshowed that a panel of markers (instead of a single marker) may beuseful to improve sensitivity and specificity for a given cell type [26].
The RP model showed that for women with intermediate-risk EC,LVSI appears particularly relevant to distinguish women with lymphnode metastases. Previous studies have demonstrated that the pres-ence of LVSI is correlated to poor prognosis tumors. Indeed, in a mul-ticenter retrospective study on type 1 FIGO stage I EC with LVSI,Simpkins et al. found an overall recurrence rate of 23%. The authors
munostaining between women with and without lymph node metastases.
Fig. 2. Optimal cut-offs denoting a correlation between estrogen and progesteron receptors semi-quantitative immunostaining and final lymph node status.
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showed that, while improving pelvic control, adjuvant radiationtherapy did not impact on recurrence rate, suggesting that systemictherapy with or without radiotherapy should be evaluated for thesepatients [27]. Moreover, in a large retrospective study on clinicopath-ological risk factors for pelvic lymph node metastases in early stageEC, Zhang et al. found that LVSI was the best criteria to predict pelviclymph node metastases with a sensitivity and specificity of 95.6% and94.5%, respectively. In addition, the authors showed that the combi-nation of LVSI and depth of myometrial invasion had a high predictivevalue with a negative predictive value and specificity reaching 97.3%and 89.1%, respectively [28]. This is quite consistent with our resultsas LVSI and depth of myometrial invasion (included in the ESMOsubgroups) were part of our predictive model, showing that thecombination of several criteria can help to better define risk subgroups.
We chose to focus on nodal status in low- and intermediate-riskEC as it currently represents a considerable matter of debate. Despitethe fact that lymphadenectomy is no longer recommended in earlystage EC since a recent meta-analysis including two randomized trialsdemonstrated no impact on survival, several discrepancies exist onthe management of patients with early stage/low- and intermediate-risk EC according to surgical staging and adjuvant therapies [29]. In arecent prospective multicenter study on the accuracy of sentinel nodebiopsy to diagnose lymph node metastases in early stage EC, Ballester
Table 1Comparison of ESMO risk group, LVSI, and estrogen/progesterone receptorsimmunostaining between patients with and without lymph node metastases.
ESMO: European Society of Medical Oncology; LVSI: lymphovascular space invasion;ER: Estrogen receptor; PR: Progesterone receptor.
et al. found that 12% of women with low- to intermediate-risk EC hadlymph node metastases that would have been overlooked if there hadbeen no lymph node staging. In this setting, the authors concludedthat SLNbiopsymay be a trade-off for these subgroups of patients to bet-ter adapt adjuvant therapies [9]. However, SLN biopsy in EC is still underassessment and there is no consensus to date on its use in routine prac-tice. In contrast, lack of information on lymph node status implies thatindications for adjuvant therapies are exclusively based on uterine find-ings. Moreover, the decision to perform systematic lymphadenectomymainly depends on primary site tumor characteristics (i.e. histologicaltype and grade, depth ofmyometrial invasion, LVSI, and cervical stromalinvolvement) [30]. Hence, the question of secondary lymphadenectomyis likely to be raised more frequently due to important discrepanciesbetween preoperative and final histology [10].
The misclassification rate of our model was 13% in the trainingdataset and 20% after bootstrap validation showing that the risk oflymph node metastases can be predicted with relatively high accuracy.This is of major importance as lymphadenectomy is not currentlyrecommended for women with early stage low- or intermediate-riskEC. Indeed, among the women without lymph node metastases thatwere predicted by the RP model, 14% had metastatic lymph nodes atfinal histology. Moreover, we found that among the women withlow-risk EC, those with ER over expression had no risk of lymph nodemetastases. In contrast, among the women with lymph node metasta-ses predicted by the RP model, only 8% had no lymph node metastasesat final histology. In addition, we found that among the women withintermediate-risk EC, those with associated LVSI and PR under expres-sion had an 88% risk of lymph node metastases. This is particularlyrelevant as secondary lymphadenectomy could be recommended forthis subgroup of patients. Indeed, several authors have suggested thatcomplete lymphadenectomymay be associatedwith improved survivaloutcomes, particularly for patientswith lymphnodemetastases [31,32].Finally, in the case of high risk of lymphnodemetastases and the impos-sibility of reoperation due to morbidity, adjuvant therapies can beadapted accordingly (e.g. external beam radiotherapy instead of vaginalbrachytherapy). In this specific setting, the PORTEC 2 trial, comparingvaginal brachytherapy (VBT) to external beam radiotherapy (EBRT) inpatients with intermediate- to high-risk EC treated without routine
Fig. 3. Recursive partitioning model illustrating histological and immunostaining profiles to predict final lymph node status.
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lymphadenectomy, reported a higher rate of pelvic recurrences after VBTcompared to EBRT suggesting that pelvic recurrences could be explainedby undetected pelvic nodal metastases that were treated by EBRT [33].
Some limits of the current study have to be underlined. First,we considered women with micrometastases and ITC as metastatic.However, the impact of micrometastases or ITC on survival is stillunder debate. Yabushita et al. found that the presence of occult me-tastases is a risk factor for recurrence in early stage EC [34]. Second,we did not assess the paraaortic lymph node status. However, previ-ous studies have shown that isolated paraaortic nodal metastasis inthe setting of negative pelvic nodes occurs in approximately 1% ofcases [35]. Third, we did not include clinical and biological parametersin themodel as no consensus exists on the relevance of such parameters(i.e. preoperative diabetes, creatinine, albumin, performance status) topredict nodal status or disease free survival. Fourth, the incidence oflymph node metastases in the low-risk group was only 9%. However,this is totally in accordance with previous studies showing that, usingultrastaging, 10% of patients with definitive low-risk endometrial can-cer have lymph node metastases, mainly related to micrometastases[9]. Finally, the high incidence of lymph node metastases in theintermediate-risk EC could be explained by the use of SLN biopsy in55% of women. Indeed, 5/16 (31%) of women with metastatic lymphnodes in the intermediate-risk EC group had micrometastases and/orITC. However, the higher the number of events, the more accurate aprediction model will be in establishing subgroups of women at risk.
Our results support that integrating immunohistochemical ER andPR profiles in a predictive model along with histological grade, depthof myometrial invasion and LVSI could help physicians to better pre-dict the final lymph node status in women with low- or intermediate-risk EC. Such tools may be helpful to better adapt surgical staging andadjuvant therapies.
Conflict of interest statement
All authors declare that they have no conflict of interest.
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