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Gynecologic Oncology
Endometrial cancer: can nodal status be predicted with curettage?$
Andrea Mariania, Thomas J. Sebob, Jerry A. Katzmannb, Patrick C. Rocheb, Gary L. Keeneyb,
Timothy G. Lesnickc, Karl C. Podratza,*
aSection of Gynecologic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USAbDepartment of Laboratory Medicine, Mayo Clinic, Rochester, MN 55905, USA
cSection of Biostatistics, Mayo Clinic, Rochester, MN 55905, USA
Received 17 March 2004
Available online 5 January 2005
Abstract
Objective. To determine whether histologic or molecular markers assessed in pretreatment curettage specimens predict nodal metastasis in
endometrial cancer.
Methods. Phenotypic and molecular variables (ploidy, proliferating cell nuclear antigen, MIB-1, p53, HER-2/neu, and bcl-2) were
analyzed in preoperative specimens from 82 patients with endometrial cancer who had lymph nodes dissected. These 82 patients had been
selected from a total population of 283 patients with endometrial cancer, using a case-cohort design. Weighted logistic regressions were then
used to determine significant predictors of positive lymph nodes, and results were estimated for the total population of 283 patients.
Results. Of the overall population, 12% of patients were estimated to have positive lymph nodes. Histologic subtype, p53, and bcl-2 each
were significantly correlated (P b 0.05) with lymph node status. With application of stepwise logistic regression, p53 was the only
independent predictor of lymph node status. In addition, a statistical model predictive of positive lymph nodes was generated which
incorporated the risk factors p53, bcl-2, and histologic subtype.
Conclusion. In pretreatment curettage specimens, the presence of unfavorable levels of p53 or bcl-2 or of nonendometrioid histologic
features, or combinations of those, significantly predicted lymph node status, thus facilitating the preoperative identification of patients at risk
of lymph node metastases.
D 2004 Elsevier Inc. All rights reserved.
Keywords: bcl-2; Dilatation and curettage; Endometrial cancer; Lymphadenectomy; Lymphatic metastases; Nonendometrioid histology; p53
Introduction
Adenocarcinoma of the endometrium is the most
common malignancy of the female genital tract in the
United States. In approximately 75% of cases, the tumor is
clinically confined to the uterus at the time of diagnosis. An
estimated 40,320 cases of endometrial cancer are expected
to be diagnosed in the United States during 2004, and an
estimated 7090 cancer deaths are expected to occur [1].
0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2004.11.030
$Presented in part at the 7th Biennial Meeting of the International
Gynecologic Cancer Society, Rome, Italy, September 26 to 30, 1999.
* Corresponding author.
E-mail address: [email protected] (K.C. Podratz).
In 1988, the International Federation of Gynecology and
Obstetrics (FIGO) suggested the incorporation of surgical
staging in the overall management of endometrial cancer
[2]. Since then, numerous studies have confirmed the
importance of pelvic and para-aortic lymphadenectomy for
defining prognosis and for providing important information
about adjuvant therapy [3]. Moreover, lymphadenectomy
may be therapeutically beneficial, at least in a subgroup of
selected patients at risk of lymph node invasion, according
to retrospective data [4,5].
Gynecologic cancer patients managed by a gynecologic
oncologist are more likely to be definitively staged
surgically than patients managed by an obstetrician-gyne-
cologist or a general surgeon [6,7]. Moreover, for the
treatment of endometrial cancer, a lymphadenectomy is less
96 (2005) 594–600
YGYNO-70695; No. of pages: 7; 4C:
A. Mariani et al. / Gynecologic Oncology 96 (2005) 594–600 595
likely to be performed in primary care hospitals than in
referral centers [8]. However, as many as 60% of patients
with endometrial cancer in the United States are treated
without the counseling of a subspecialty-trained physician
[9]. The ability to predict lymph node invasion preoper-
atively would potentially enable primary care physicians to
refer high-risk patients to subspecialty-trained physicians
who had the surgical expertise necessary for proper staging,
with the possibility of enrollment in new treatment protocols
for preventing lymphatic recurrences [10]. Unfortunately,
preoperative clinical staging of endometrial cancer fails to
detect the presence of extrauterine disease in 22% of
patients presumed to have tumor confined to the uterus [11].
Different cytokinetic [12] and molecular [13] events
measured in hysterectomy specimens of patients with
endometrial cancer have been shown to be associated with
lymph node metastases. We previously demonstrated that
the molecular and cytokinetic analysis of preoperative
endometrial tissues may enable the prediction of prognosis
and extrauterine disease before primary therapy [14]. In the
present study, we tested the hypothesis that molecular and
cytokinetic parameters, measured in pretreatment endome-
trial samples, are predictive of lymph node invasion in
patients with corpus cancer.
Patients and methods
From 1984 through 1993, 815 patients with surgically
treated endometrial cancer were accrued to the database at
Mayo Clinic (Rochester, MN). We reviewed records of 299
patients with epithelial endometrial cancer who satisfied the
following inclusion criteria: (1) their cancer had been
surgically managed with hysterectomy and removal of
remaining adnexal structures, (2) another malignancy had
not been diagnosed within 5 years before or after the
diagnosis of endometrial cancer (with the exception of
patients with carcinoma in situ or with skin cancer other
than melanoma), and (3) they had both initial preoperative
endometrial sampling and definitive surgery at our institu-
tion. Sixteen of the 299 patients had insufficient clinicopa-
thologic data or tissue blocks available; thus, a cohort of 283
patients remained. From the subgroup of 283 women, 125
were selected using a case-cohort design. This group of 125
women included all 49 women experiencing a recurrence
and 76 randomly chosen progression-free patients. This was
not a random sample of the whole cohort, but the selection
was done in accord with the design of Prentice [15]. We
assumed that the randomly selected subgroup of 76
nonrecurrent cases was representative of the whole pop-
ulation of the 234 recurrence-free patients. The present
analysis addresses the 82 patients (66%), from the selected
125, who had lymph nodes dissected (34 who had
recurrences and 48 who were recurrence-free).
Hematoxylin–eosin-stained slides of the preoperative
endometrial tissue samples from the 82 selected cases were
reviewed by two of us (T.J.S. and G.L.K.), who confirmed
the histologic diagnosis, assessed the tumor grade and
subtype, and selected the appropriate area of the tumor from
which to study the cytokinetic and molecular parameters.
Staging was defined according to the FIGO classification
[2]. In cases with operations before 1988, the stage was
determined retrospectively on the basis of operative and
pathology reports. Histologic classification was performed
according to the World Health Organization classification
[16]. Architectural grading was based on the degree of
glandular differentiation in accordance with the FIGO
guidelines [2].
A gynecologic oncologist was responsible for all surgical
staging procedures. Surgical staging included palpating all
abdominal organs and obtaining peritoneal washings for
cytologic evaluation. In the absence of macroscopic
metastases, total hysterectomy with removal of adnexal
structures was performed. When indicated, additional
surgical procedures included omentectomy, appendectomy,
and cytoreductive procedures. Frozen section examination,
including intraoperative assessment of hysterectomy speci-
mens and all lymph nodes, was routinely available for all
cases. Therefore, the need for and the extent of the lymph
node dissection were primarily dictated by the intraoperative
assessment of frozen sections for histologic grade, depth of
invasion, and presence of extrauterine disease. As a general
rule, patients with low-grade disease and superficial
myometrial invasion were less likely to have a lymphade-
nectomy than patients with more aggressive disease.
However, the surgical procedures were performed during a
10-year period primarily by five gynecologic oncologists at
our institution, and the criteria for performing a lymphade-
nectomy and its actual extension varied according to the
time and the surgeon.
Analysis of cytokinetic and molecular variables
The analysis of cytokinetic and molecular variables in
endometrial cancer tissues has been described previously
[14]. Briefly, the immunohistochemical staining technique
used a modification of the avidin–biotin method reported by
Hsu et al. [17]. The paraffin blocks were cut in 6-Am sections
and mounted on silanized glass slides. The slides were
dewaxed. Endogenous peroxidase activity was blocked, all
sections were subjected to heat-induced epitope retrieval,
and nonspecific binding sites were blocked. Automated
immunohistochemical staining was performed with a Tech-
Mate 500 (Ventana Medical Systems, Tucson, AZ), and
detection reagents were obtained from Ventana Medical
Systems and used according to the manufacturer’s instruc-
tions. Staining was performed with primary antibodies
against proliferating cell nuclear antigen (PCNA) (clone
PC10 [DAKO Corp., Carpinteria, CA], diluted 1:7500 in 1%
normal goat serum/phosphate-buffered saline/Tween-20),
p53 antigen (clone DO-7 [DAKO Corp.], diluted 1:200),
HER-2 protein (A0485 [DAKO Corp.], diluted 1:1600), Ki-
A. Mariani et al. / Gynecologic Oncology 96 (2005) 594–600596
67 antigen (clone MIB-1 [Immunotech, Westbrook, ME],
diluted 1:100), and bcl-2 oncoprotein (M0887 [DAKO
Corp.], diluted 1:25).
Nuclear staining for p53, PCNA, and MIB-1 was
quantitated on a CAS 200 Image Analysis System (Bacus
Laboratories, Inc., Lombard, IL) by the Quantitative
Proliferation Index program. Cell nuclei were analyzed by
two image acquisition cameras that measure absorption at
wavelengths of 500 and 620 nm. All nuclei were measured
at 620 nm, but only the diaminobenzidine (DAB)-positive
nuclei were measured at 500 nm.
The proliferating index or percentage nuclear staining
was calculated by dividing the summed DAB-positive
nuclear areas detected at 500 nm by the summed optical
density measured at 620 nm. At least thirty fields were
quantified per case using a 40� lens (total magnification
400�). While the areas measured were collected in a
random, yet systematic manner, in cases of focal, low
expression, cells with nuclear staining were always included
in the analysis. Antigen levels were then expressed as a
percentage of the stained area (a continuous variable from
0% to 100%) by using the instrument to compare the
summed optical density of DAB with hematoxylin.
Grading of the slides for the 2 markers bcl-2 and HER-2/
neu included glandular staining intensity relative to the
corresponding negative control slide. The reviewers
assigned the following scores: 0, no staining; 1+, weak
staining; 2+, moderate staining; and 3+, strong staining.
After the quantitation of nuclear and cytoplasmic
markers, one of us (T.J.S.) subjectively assessed the slides
for their adequacy and preservation. Cases were eliminated
from the analysis if the tissue had poor preservation that
made the staining uninterpretable or if there was no residual
tumor tissue on the slide.
Analysis of DNA content
The analysis of DNA content of endometrial cancer tissues
was conducted as previously reported [18]. Briefly, nuclear
suspensions were prepared from paraffin-embedded tissue
blocks with the technique described by Hedley et al. [19] after
histologic documentation of tumor adequacy. The paraffin
from 3 or more 50-Am sections cut with a standard microtome
was solubilized with Histo-Clear (National Diagnostics,
Atlanta, GA). The tissue was rehydrated, incubated (2.5 h
at 378C) in a 0.5% pepsin solution (P7012, Sigma, St. Louis,
MO), and filtered and centrifuged at 100 g for 15 min. The
resuspended pellet was treated with a citrate-buffered trypsin
inhibitor (T9253, Sigma) solution for 10 min and with
ribonuclease A (R4875, Sigma) for an additional 30 min. The
isolated nuclei were resuspended and treated with propidium
iodide (P5264, Sigma) for a minimum of 1 h with the method
described by Vindelov et al. [20]. The resulting suspension
was sonicated as described by Gonchoroff et al. [21].
Nuclear content was measured on a Cytoron Absolute
flow cytometer (Ortho Diagnostic Systems, Inc., Raritan,
NJ). Specimens were standardized with a normal kidney
control sample and the instrument was set to channel 50.
Histograms of 10,000 nuclei were recorded for each
specimen at a maximal scanning flow rate of 1000 nuclei
per second. Cell cycle evaluation of the DNA histograms
derived from flow cytometry was performed with ModFit
software (version 5.2; Verity Software House Inc., Topsham,
ME). Data acquisition was triggered on red fluorescence and
the cell cycle distribution was determined by analyzing
ungated data from 10,000 nuclei in a rectangular S-phase
computer model [22]. Tumors with only one G0/G1 peak
were designated as diploid (i.e., 2n), whereas tumors with
histograms suggesting more than one G0/G1 population
were categorized as aneuploid. Tumor samples with at least
9% of nuclei associated with the 4n peak were considered
tetraploid. Specimens showing at least 3 G0/G1 peaks were
classified as multiploid but were analyzed collectively with
the aneuploid tumors.
The DNA index (DI) was calculated as the ratio of the
mean channel of the aneuploid G0/G1 population to the
mean channel of the diploid G0/G1. Therefore, diploid
tumors had a DI of 1.0; tetraploid, 2.0; and aneuploid,
greater than 1.0 and less than 2.0. The most prominent
population in multiploid tumors was used to assign a DI.
The proliferative index (PI) was defined as the sum of the
percentage of cells in the S-phase fraction (SPF) and the
percentage of cells in G2/metaphase.
Cases were considered unsuitable for analysis if the
endometrial sample did not have enough cells, if no tumor
was detected in the slide, if the histogram was not
interpretable, or if the coefficient of variation was greater
than 10.
Statistical analysis
For the statistical analyses, the cytokinetic and molecular
variables were dichotomized. The quantitative DNA varia-
bles were divided with the same cutoffs we used previously
[14,23]. Patients with diploid tumors were compared with
patients who had aneuploid/tetraploid tumors. We compared
patients who had a DI b 1.5 with patients who had a DI z1.5; patients who had an SPF b 9% with those who had an
SPF z 9%; and patients who had a PI b 14% with those
who had a PI z 14%.
On the basis of our previous experience [14] and the
distribution of our sample, we divided p53, MIB-1, and
PCNA at 33% and compared patients with no staining or
weak staining (V33%) with patients who had moderate or
strong staining (N33%).
HER-2/neu and bcl-2, quantitated by the pathologist
(T.J.S.) as categorical variables, were divided as follows:
patients with strong staining for HER-2/neu (3+) were
compared with the others (i.e., those with no staining, 1+, or
2+). Moreover, patients not expressing bcl-2 were compared
with patients who had weak, moderate, or strong (i.e., 1+,
2+, or 3+) cytoplasmic staining for bcl-2. This was done as
A. Mariani et al. / Gynecologic Oncology 96 (2005) 594–600 597
in our previous study [14]. For traditional histologic
variables, patients with histologic grades 1 and 2 were
compared with those with grade 3; furthermore, patients
with endometrioid tumors were compared with patients who
had a nonendometrioid histologic subtype (i.e., serous, clear
cell, undifferentiated, mucinous).
Weighted logistic regressions and contingency tables
were used to model lymph node status [24]. Owing to the
fact that the actual study population was composed of 82
patients, but that the final results were a weighted estimation
for the overall population of 283 patients, the results were
usually reported as percentages and not as raw numbers.
Differences between groups were considered statistically
significant at P b 0.05. SAS version 6.12 (SAS Institute
Inc., Cary, N.C.) and S-PLUS version 3.4 (MathSoft, Inc.,
Seattle, WA) statistical software packages were used.
Table 1
Prediction of lymph nodes positive for disease (pelvic or para-aortic or
both) by using single variables measured in preoperative endometrial
samples
Variable Patient, %a PLN, %a,b Pc ORd
Histologic grade 0.52 1.38
1–2 71 11
3 29 14
Histologic subtype 0.02 3.23
Endometrioid 80 9
Nonendometrioid 20 24
p53 0.003 5.77
V33% 90 9
N33% 10 37
MIB-1 0.36 1.71
V33% 29 8
N33% 71 13
PCNA 0.98 1.01
V33% 36 12
N33% 64 12
bcl-2 0.02 0.33
0 28 22
1+, 2+, or 3+ 72 8
HER-2/neu 0.40 1.79
0, 1+, or 2+ 90 11
3+ 10 18
Ploidy 0.98 1.02
Diploid 74 12
Aneuploid or tetraploid 26 12
SPF 0.28 1.71
b9% 69 10
z9% 31 16
PI 0.73 1.18
b14% 51 11
z14% 49 13
DI 0.47 0.62
b1.5 78 13
z1.5 22 8
DI, DNA index; OR, odds ratio; PCNA, proliferating cell nuclear antigen;
PI, proliferative index; PLN, positive lymph node; SPF, S-phase fraction.a Estimated for the overall population of 283 patients, considering the 82
patients who had lymph nodes dissected.b Estimated probability of patients having PLNs.c Wald test in weighted logistic regression.d OR from weighted logistic regression model.
Results
The mean age (FSD) of the 82 sampled patients was
63.2 F 9.5 years (range, 40–87 years) and the mean body
mass index (FSD) was 29.3 F 8.1 (range, 17.6–57.6). In
three patients, distant metastases were detected before
treatment: one patient had metastases in the liver, one in
the lung, and one in the bones. All 82 patients had
hysterectomy and bilateral salpingo-oophorectomy associ-
ated with pelvic or para-aortic lymphadenectomy or both.
More precisely, 56 patients had pelvic lymphadenectomy
only, 24 had both pelvic and para-aortic lymphadenectomy,
and 2 had para-aortic lymphadenectomy only. The median
number of pelvic lymph nodes dissected was 15 (range, 1–
55), whereas the median number of para-aortic lymph
nodes removed was 4.6 (range, 1–13). Overall, 58 of the
82 patients (71%) had more than 10, and 25 of them
(30%) had more than 20 pelvic lymph nodes dissected.
Moreover, 15 of the 82 patients (18%) had lymph nodes
positive for disease (bpositive nodesQ). More precisely, all
15 patients had positive pelvic lymph nodes, and 5 of them
had associated positive para-aortic lymph nodes. No
patient had positive para-aortic and negative pelvic lymph
nodes.
During review of the immunohistochemically stained
slides, six cases were eliminated because of technical
inadequacies. Thus, the analysis of molecular and traditional
markers was conducted on 76 cases. For the quantitative
DNA analysis, two cases had insufficient tissue, and six
had a coefficient of variation greater than 10. Moreover,
one other case was not interpretable for the SPF and the PI.
Prediction of lymph nodes positive for disease (pelvic or
para-aortic or both)
Of the overall population of 283 patients, 12% were
estimated to have positive lymph nodes. Significant associ-
ations were detected between positive nodes and each of the
following, measured on the preoperative endometrial sam-
ple: p53 (odds ratio [OR] = 5.77; P = 0.003), bcl-2 (OR =
0.33; P = 0.02), and histologic subtype (OR = 3.23; P =
0.02). MIB-1, PCNA, HER-2/neu, histologic grade, ploidy,
SPF, PI, and DI did not significantly distinguish between
patients with positive nodes and patients without lymph node
invasion (Table 1).
A stepwise weighted logistic regression model, including
all the significant predictors of positive nodes, showed that
only p53 was independently associated with lymph node
positivity (OR = 5.35; P = 0.003).
We generated a statistical model predictive of positive
nodes by using a combination of the three variables that
were significantly associated with lymph node positivity in
the univariate analysis (i.e., p53, histologic subtype, and
A. Mariani et al. / Gynecologic Oncology 96 (2005) 594–600598
bcl-2). The baseline estimated probability of positive nodes
was 6%. When one of the variables differed from the
baseline values, the estimated probability increased to a
percentage between 13% and 17%; when two variables
differed, the estimated probability increased to a value
between 28% and 34%; and when three variables differed,
to 55% (Table 2). The positive predictive value (PPV) of the
presence of at least 1 high-risk variable was 26%, with a
negative predictive value (NPV) of 94%.
Considering only those variables traditionally measured
in preoperative samples, we observed a 10% probability of
positive lymph nodes occurring in patients with grade 1 or
grade 2 endometrioid tumor, compared with 17% in patients
with grade 3 tumor and/or nonendometrioid cancer. The
PPV of the above combined traditional predictors was 17%,
with an NPV of 90%.
Among patients traditionally considered at low risk of
lymph node invasion (i.e., endometrioid grade 1 or 2), those
who had at least one of the two molecular variables
predictive of positive lymph nodes (i.e., negative bcl-2 or
positive p53) had a 16% probability of having lymph node
metastases, compared with 7% among patients with non-
predictive molecular markers.
Discussion
Since 1988, surgery has been considered the primary
treatment of endometrial cancer [2]. In fact, complete
surgical staging may provide detailed prognostic informa-
tion, thus guiding the postoperative management of patients
[3]. Moreover, surgical staging may possibly have ther-
apeutic value [4,5]. However, most patients with endome-
trial cancer in the United States are treated surgically by
physicians managing a very limited number (b5) of such
Table 2
Logistic regression model predicting lymph node involvement through
measurement of p53, histologic subtype, and bcl-2 in preoperative endo-
metrial samples, assuming an additive effect of the 3 markers ( P = 0.005)
bcl-2 Histologic
subtype
p53, %a Patients, %b ORc PLN, %b,c,d
Pos Endo V33 58.8 1 6
Pos Nonendo V33 7.9 2.41 13
Neg Endo V33 16.5 2.53 14
Pos Endo N33 0.1 3.22 17
Neg Nonendo V33 6.2 6.08 28
Pos Nonendo N33 5.5 7.75 33
Neg Endo N33 1.5 8.14 34
Neg Nonendo N33 3.5 19.58 55
Endo, endometrioid; Neg, negative; Nonendo, nonendometrioid; OR, odds
ratio; PLN, lymph node positive for disease; Pos, positive.a p53 V 33%, no staining or weak staining; p53 N 33%, moderate or strong
staining.b Estimated for the entire population of 283 patients.c OR and estimated PLN from weighted logistic regression model.d Estimated probability of patients having PLNs.
cases annually [25]. Typically, these physicians lack detailed
knowledge of the natural history of the disease and of the
indications for adjuvant treatment at the time of surgery as
well as the intraoperative expertise to perform pelvic and
para-aortic lymphadenectomy and cytoreduction as indi-
cated; consequently, patients frequently receive therapy that
is substandard for optimal outcomes. Therefore, the
preoperative identification of patients who have endometrial
cancer and who are at risk of lymph node involvement
would allow the referral of high-risk patients to tertiary care
centers or the participation of gynecologic oncologists in the
overall management, thus increasing the likelihood that
patients would be treated by physicians who had the
appropriate level of clinical and surgical expertise [8].
At present, in patients with endometrial cancer, the
preoperative assessment and prediction of lymph node
involvement is inaccurate [11]. On the basis of histologic
assessment of hysterectomy specimens, three main uterine
variables are highly predictive of lymph node metastasis:
deep myometrial invasion, histologic grade, and cervical
involvement [11]. Therefore, these three variables would be
potentially useful for the preoperative selection of patients at
risk of lymphatic invasion. Numerous reports have focused
on the preoperative assessment of the depth of myometrial
invasion and cervical involvement by use of imaging
techniques [26,27], serum CA-125 levels [18,28], cervical
cytology [27,29], and hysteroscopy [27], with various,
sometimes encouraging, results. However, tumor grade in
preoperative endometrial sampling is not sufficiently accu-
rate for selecting patients at risk. In fact, 17% of patients
with histologic grade 1 present with deep myometrial
invasion; moreover, nearly 20% of the endometrial tumors
are upgraded after review of permanent sections of
hysterectomy specimens [30]. Furthermore, the assessment
of grade in the preoperative biopsy is somewhat subjective
and is associated with considerable interobserver variability
[31]. Potentially, the use of tumor markers amenable to
automated quantitation would minimize the diagnostic
subjectivity observed with traditional histologic assessment
[32]. In addition, a panel of prognostic factors may enhance
the diagnostic accuracy in detecting patients who are at risk
of extrauterine disease [14]. Thus, in the present study, we
analyzed preoperative endometrial cancer specimens by use
of a series of cytokinetic and molecular variables that have
been previously demonstrated to be associated with lymph
node metastases [13].
Ploidy and cytokinetic parameters determined by flow
cytometry are recognized prognostic factors in endometrial
cancer and appear to effectively predict advanced disease
[12,14]. Nevertheless, in our population, we observed that
none of the flow cytometric variables was significantly
associated with the presence of positive lymph nodes
(Table 1).
Reports of the use of MIB-1/Ki-67 and PCNA to predict
extrauterine disease are conflicting [33,34]. Furthermore, in
various studies there was a lack of association of HER-2/neu
Table 3
Prediction of lymph nodes positive for disease (pelvic or para-aortic or
both) in patients with endometrial cancer according to assessment of
various markers in pretreatment curettage specimens versus hysterectomy
specimens
Study Specimen Marker Measure PPV
(%)
NPV
(%)
Present
study
Curettage p53, bcl-2, HS 0 vs. 1
or more
26 94
Grade 3, HS 0 vs. 1
or more
17 90
Creasman
et al. [11]aHysterectomy Grade 1–2 vs. 3 18 94
Myometrial
invasion
Inner
third vs.
outer two
thirds
16 96
HS, histologic subtype; NPV, negative predictive value; PPV, positive
predictive value.a Prediction for positive pelvic lymph nodes.
A. Mariani et al. / Gynecologic Oncology 96 (2005) 594–600 599
overexpression with the stage of disease [14,35]. In our study,
MIB-1, PCNA, and HER-2/neu overexpressions were not
significantly associated with positive lymph nodes (Table 1).
However, reports have suggested that p53 overexpression
[36] and the lack of expression of bcl-2 [13] in hysterectomy
specimens are associated with extrauterine disease. Sim-
ilarly, we observed that p53 overexpression and the absence
of detectable levels of bcl-2 were predictive of lymph node
involvement (Table 1). Through stepwise logistic regres-
sion, p53 was the only independent variable associated with
positive lymph nodes, thus permitting the discrimination of
a small subgroup of patients who are at high risk of
lymphatic metastases. This finding is in accord with our
previous observation that p53 overexpression, measured in
hysterectomy specimens, is a strong and independent
predictor of distant failures in endometrial cancer [36].
A model using all three variables that significantly
predicted lymph node invasion (i.e., p53, bcl-2, and
histologic subtype) allowed us to estimate the probability
of patients having positive lymph nodes by using different
combinations of the preoperative markers (Table 2). When
none of the three variables was abnormal, the estimated
probability of finding positive lymph nodes was 6%, and
when only one was aberrant, the probability ranged from
13% to 17%, whereas when all 3 variables were abnormal,
the probability of finding positive lymph nodes was 55%
(Table 2). Therefore, the PPV in the presence of at least 1
high-risk variable was 26%, with an NPV of 94%. This is a
slight improvement when compared with the PPV and NPV
achieved using only the traditional preoperative variables
grade and histologic subtype (Table 3). However, it is
noteworthy that when considering only patients traditionally
considered at low risk of lymph node invasion (i.e.,
endometrioid grades 1 and 2), the molecular markers bcl-2
and p53 further separated patients on the basis of their risk
of having positive lymph nodes. In fact, among low-risk
patients, we estimated that 16% would have positive lymph
nodes when at least one of the two molecular variables was
predictive of lymph node invasion, compared with 7% in
patients with nonpredictive molecular markers.
Review of historical data shows that histologic grade,
measured at the time of hysterectomy, has a PPVof 18% and
an NPVof 94% for predicting lymph node status; moreover,
depth of myometrial invasion has a PPV of 16% and an
NPV of 96% [11] (Table 3). Therefore, the histologic and
molecular variables, as measured in the preoperative
endometrial curettage in the present study, predict lymph
node status similarly to the traditional histopathologic
variables identified in hysterectomy specimens. However,
even our molecular assessment of preoperative predictors of
lymph node invasion remains imprecise.
A possible limitation of the present analysis is the
relatively small number of patients in our population who
were actually analyzed with the prognostic markers (n =
82), and consequently, the relatively limited number of
patients with positive lymph nodes (n = 15). Moreover,
owing to the weighted statistical design, the study was based
on the assumption that patients who were free of recurrence
and who were analyzed (n = 48) were representative of the
other 186 nonrecurrent cases, from the total population of
283 patients with endometrial cancer.
Preoperative molecular staging will require that paraffin-
embedded tissue of patients diagnosed with endometrial
cancer in community hospitals to be sent to tertiary care
centers for immunohistochemical analysis. The immunohis-
tochemical quantitation may be performed in less than 72 h
and with very affordable costs. A necessary requirement for
the practical usefulness of preoperative molecular staging is
that physicians working in community hospitals would be
willing to select and refer high-risk endometrial cancer
patients to tertiary care centers for surgical staging.
Our study suggests that the preoperative evaluation of
quantifiable molecular variables aids in selecting patients
who are (or are not) at risk of lymph node invasion. The
preoperative assessment of p53 expression, histologic
subtype, and bcl-2 identified a subgroup (41% of the overall
population) of patients with endometrial cancer who had a
risk of at least 13% (mean, 20.6%) of having positive lymph
nodes (Tables 2 and 3). Potentially, these patients would in
turn benefit from referral to tertiary care centers or from the
preoperative counseling of a gynecologic oncologist. As the
pathogenesis is further defined, we are optimistic that
molecular staging criteria will afford physicians the oppor-
tunity to distinguish patients at risk of extrauterine disease
before initiating therapy.
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