ANATOMIC PATHOLOGY
Original Article
Flow Cytometric Analysis of Endometrial
Stromal Sarcoma
CHARLES L. HITCHCOCK, M.D., P H . D . / AND HENRY J. NORRIS, M.D. 2
Twenty-two endometrial stromal sarcomas were studied by flow
cytometric analysis and the results were correlated with surgical
stage, nuclear grade, mitotic index, and recurrence. Ploidy determination was not helpful in predicting recurrence in patients
with Stage I disease because all 14 were diploid. Only 2 of the
22 tumors were aneuploid; both were high-stage neoplasms. Cell
proliferation (%S or %S + %G2/M) did not significantly correlate
Endometrial stromal sarcomas (ESS) are rare tumors
composed of cells closely resembling the stromal cells of
a proliferating endometrium.1,2 Low-grade and high-grade
ESS are distinguished on the basis of mitotic rate2 or degree
of nuclear atypia.3 Endometrial stromal sarcoma has the
propensity for indolent growth, yet one third of neoplasms
initially confined to the uterus (Stage I) recur.
We designed this study to determine if either DNA
ploidy or cell proliferation correlated with the pathologic
features and the clinical behavior of ESS. Flow cytometric
analysis of archival material from 22 patients with ESS
was performed and the results were compared with the
stage, degree of atypia, mitotic index, and incidence of
tumor recurrence of Stage I disease.
with vascular invasion, nuclear grade, mitotic index, or surgical
stage. Of the 13 Stage I neoplasms with follow-up data, three
recurred. The mean proliferation index (%S + %G2/M) of those
that recurred was 12.81 ± 0.47, which did not differ from those
that did not recur (mean 12.25 ± 4.11). (Key words: Endometrial
stromal sarcoma; Flow Cytometry; DNA; Ploidy) Am J Clin
Pathol 1992; 97:267-271
(Table 1). All neoplasms had infiltrating margins and
contained uniform cells resembling proliferative-phase
endometrium. We stratified cases according to surgical
stage, nuclear atypia, as previously defined,2'4 and mitotic
counts. The highest mitotic count in 10 consecutive contiguous high-power fields was recorded. A high-power field
was 0.156 mm 2 . We excluded from the study those neoplasms with marked atypia because they were of uncertain
lineage. Thus the maximum atypia was 2+ and reflected
some hyperchromatism, nuclear enlargement, prominent
nucleoli, and clumping of chromatin about the nuclear
membrane (Fig. 1). Patient information and tumor characteristics were obtained from Armed Forces Institute of
Pathology records and from questionnaires mailed to the
patient's referring physician.
MATERIALS AND METHODS
Paraffin sections were prepared for DNA staining and
flow cytometric analysis, as previously detailed.5 Briefly,
Paraffin blocks of 22 cases of ESS were selected from
nuclei were isolated from two or three 80-/im-thick secthe files of the Armed Forces Institute of Pathology and
tions. We avoided necrotic, fibrous, and hemorrhagic areas
used for this study set. We reviewed hematoxylin-andby scoring the block face and isolating the desired regions
eosin-stained sections from each block and analyzed them
from the sections. Two or more paraffin blocks were samfor mitotic activity, cellular atypia, and vascular invasion
pled from 16 of 22 cases. The paraffin-embedded sections
were deparaffinized, rehydrated, washed in two changes
of phosphate-buffered saline, and stored overnight at 4
2
From the 'Departments of Cellular Pathology and Gynecologic and °C. Enzymatic digestion was performed in 0.25% pepsin
(P-6887, Sigma Chemical Co., St. Louis, MO) for 90 minBreast Pathology, Armed Forces Institute ofPathology, Washington, D.C.
utes at 37 °C. The isolated nuclei were counted in a CoulThe opinions and assertions contained herein do not purport to be
ter
ZM Cell Counter (Coulter Electronics, Hialeah, FL)
official or represent the views of the Departments of Air Force or Defense.
and stained with propidium iodide (Calbiochem-Boehring
Received March 6, 1991; received revised manuscript and accepted
for publication June 6, 1991.
Diagnostics, San Diego, CA; 50 fig/mL phosphate-buffAddress reprint requests to Dr. Norris: Department of Gynecology/
ered
saline/106 nuclei) as previously described.5 The
Breast Pathology, Armed Forces Institute of Pathology, Washington,
sample was aspirated repeatedly through a 23- to 26-gauge
DC 20306-6000.
267
268
ANATOMIC PATHOLOGY
Original Article
TABLE 1. CLINICAL FEATURES OF ENDOMETRIAL STROMAL SARCOMAS
Case
Age (yrs)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
50
49
41
49
54
48
36
42
36
45
46
38
28
41
47
38
42
27
44
59
45
65
Stage
Vascular
Invasion
MI
Cell Atypia
6
' 4
6
1
0
1
7
0
4
13
1
30
20
2
17
2
2
10
4
30
10
4
0
1+
1+
1+
0
0
0
0
1+
1+
0
II
II
II
III
III
IV
IV
IV
No
No
No
No
No
Yes
No
Yes
Yes
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
1+
0
1+
0
0
1+
0
1+
2+
1+
0
Recurring/
Persistent
No
No
No
No
No
No
No
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
DNAAP
F/U (yrs)
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
No
AW 9.9
AW 16.8
AW 15.1
AW 12.0
AW 10.2
AW 10.1
AW 21.8
AW 10.0
AW 26.0
Lost
AW 9.7
AW 12.0
AT 12.7
AT 18.0
Lost
DT 14.8
AT 8.7
AT 7.5
AW 3.5
DT 2.0
DT 0.6
AW 4.4
Ml = number of mitotic figures per 10 high-power fields; AP = DNA aneuploid; F/U = follow-up; AW alive and well; AT = alive with tumor, DT = dead with tumor.
needle and filtered through 37-fitn nylon mesh (TEKO,
Inc., Elmsford, NY) just before flow cytometric analysis.
The DNA content of the isolated nuclei was measured
on an EPIC 541 flow cytometer (Coulter Electronics), as
previously described.5 We analyzed a minimum of 2
X 104 nuclei per sample (1-2 X 106 nuclei/mL) at a flow
rate of no more than 100 events per second. Debris and
doublets were minimized by gating on peak and integrated
red signals.
Samples were analyzed and classified without knowledge of the patients' clinical course. We classified tumors
as DNA-diploid (a single GO/Gl peak) or DNA-aneuploid
(a distinct second GO/Gl peak). We used a model, based
on multiple broadened rectangles (DNA SFIT method 4
CytoLogic program V2.1, Coulter Electronics, Hialeah,
FL) for analysis of DNA histograms. The DNA histogram
with the lowest full-height coefficient of variation (CV) of
the prominent G0/G1 peak was used for subsequent cellcycle analysis. Debris was subtracted by using a "split
nuclei" algorithm. We combined the percentage of cells
in the S and G2/M phases of the cell cycle to form a cell
proliferation index (PI). Statistical analysis of the data
used the Student's Mest and Fisher's exact test. The results
are given as the mean ± standard deviation and probability
values.
RESULTS
Analyzable DNA histograms were obtained from all
but 4 of the 43 blocks studied (Fig. 2). The coefficient of
variation (CV) of the GO/Gl peak ranged from 3.76 to
14.74 (mean, 6.66 ± 1.93) for these 39 histograms. We
excluded DNA histograms with a CV greater than 10.5
from further analysis. Usable data was obtained from all
cases except case 13. For those cases with two or more
blocks, we used the DNA histogram with the lowest CV
for analysis. Of the 21 DNA histograms analyzed, the CV
ranged from 3.76 to 10.42 (mean, 6.11 ± 1.38).
Only two ESS (cases 20 and 21) exhibited a DNAaneuploid cell population. Both were high-grade, Stage
IV lesions with cellular atypia. Analysis of two blocks from
these cases indicated that one tumor (case 20) exhibited
two separate DNA-aneuploid stemlines (DI, 1.46 and
1.64). No other tumors exhibited multiple DNA-aneuploid populations.
We measured cell proliferation as the percentage of cells
in the S-phase fraction and as a proliferation index (%S
+ G2/M fractions) (Table 2). The S-phase fraction ranged
from 0.64 to 14.6% (mean, 5.34 ± 3.62%). The mean PI
was 12.06 ± 5.49%, ranging from 5.42 to 31.45%. The
two DNA-aneuploid (cases 20 and 21) had Pis of 21.27%
and 25.84%, respectively. Heterogeneity in the PI was
noted in those cases in which multiple blocks were sampled. Differences ranged from 0.11 to 13.48% (mean, 3.17
± 3.32%). There was no significant increase in the Pis (Sphase fraction data not shown) in the 15 tumors with a
high mitotic index as compared to the 6 ESS with low
mitotic index (P = 0.705) (Table 2). There was no statistically significant correlation (P = 0.474) between the de-
A.J.C.P. • February 1992
HITCHCOCK AND NORRIS
Flow Cytometric Analysis of Endometrial Stromal Sarcoma
269
FIGS. 1A and B. (A) DNA diploid Stage 1 endometrial stromal sarcoma (case 12) that did not recurr. (B) DNA aneuploid Stage IV endometrial
stromal sarcoma (case 20) that did recur.
gree of cellular atypia and cell proliferation. The PI was
11.00 ± 2.12 in the 10 cases without atypia compared to
12.84 ± 6.45 in the 11 cases with cellular atypia. Stage
II-IV ESS lesions exhibited increased cell proliferation,
although not significantly (P = 0.102), as compared to
Stage I neoplasms. In addition, the mean PI of Stage I
ESS lesions that recurred was 12.81 ± 0.47, but it did not
differ significantly from the PI of those that did not recur
(mean, 12.25 ±4.11).
DISCUSSION
Histologic features alone are not satisfactory indicators
of the prognosis of patients with ESS.2,4,6"8 Chang and
associates,4 in a recent report of 109 patients with ESS,
used mitotic activity2 and cellular differentiation3 to divide
ESS into low and high grades. They concluded that tumor
stage rather than grade is the most powerful predictor of
prognosis. Our study set was too small and the follow-up
too short (only one patient died despite a follow-up period
of up to 26 years) for adequate testing of this point.
Our results from flow cytometric analyses of archival
material are consistent with reports using image analysis
of Feulgen staining9 and flow cytometry. 10 " Endometrial
stromal sarcoma lesions with less than 10 mitotic figures
per 10 high-power fields had diploid DNA contents. In
contrast, we found that ESS with more than 10 mitotic
figures per 10 high-power fields had variable DNA contents (Table 2). Two of eight (25%) of these samples contained a DNA-aneuploid cell population and exhibited
increased mitotic activity and cellular atypia, and both
patients died of tumor. August and colleagues10 reported
that four of five (80%) high-grade neoplasms, defined microscopically using Evan's criteria,3 had DNA-aneuploid
cell populations. DNA-aneuploidy also has been reported
in one of two recurrent lesions." Yet these studies are too
small to define the clinical significance of DNA-ploidy in
ESS. Judging from our results, ploidy lacks significance
relative to mitotic activity, cellular atypia, stage, or tumor
recurrence.
Cell proliferation, as measured by flow cytometry, is a
significant indicator of clinical behavior for several tumors.12"16 The clinical significance of this parameter has
been simplified by the introduction of modeling algorithms to minimize the impact of debris on DNA histograms.17 These programs make it possible to quantitate
cell-cycle parameters more accurately. Low cell proliferation (%S < 10%) has been reported to be a feature of
Vol. 97 . No. 2
270
ANATOMIC PATHOLOGY
Original Article
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30
60
90
120
150
180
210
240
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FIG. 2 Examples of DNA histograms. (A)
Diploid DNA histogram from low-grade,
Stage I nonrecurring endometrial stromal
sarcoma (CV = 5.80, % S = 4.00). (B) Aneuploid DNA histogram from Stage IV endometrial stromal sarcoma (Aneuploid: G0/G1;
DI, 1.63, CV = 5.66, % S = 8.99).
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DNA-diploid ESS, whereas DNA-aneuploid ESS have cell
proliferation values of up to 34%.10 The number of cases
was too small to correlate these results with tumor stage
or recurrence. Our results demonstrate that cell proliferation (%S + %G2/M) is increased in some lesions with
increased mitotic activity, vascular invasion, recurrence,
and increased surgical stage (II-IV). These results are
similar to those recently reported by August and colleagues,10 although the relationships lack statistical significance due, in part, to thefindingthat cell proliferation
varies among blocks from the same neoplasm.
Since its introduction by Hedley,16 flow cytometric
analysis of paraffin-embedded sections has been used to
study an ever-increasing number of tumors. This study
demonstrates further the advantage of flow cytometric
analysis of archival material. The technique allows rapid
study of rare neoplasms, such as ESS, that have known
follow-up data. However, flow cytometric analysis does
not always provide clinically relevant data. Although we
found that DNA-aneuploidy and increased cell proliferation are common in high-stage and recurrent lesions, the
association is not statistically significant.
A.J.C.P. • February 1992
HITCHCOCK AND NORRIS
Flow Cytometric Analysis of Endometrial Stromal Sarcoma
TABLE 2. FLOW CYTOMETRIC FEATURES OF
ENDOMETRIAL STROMAL SARCOMA
DNA Ploidy
Proliferation Index
Tumor
Features
Cases
Mitotic Activity
<10/10HPF
&10/10HPF
Cellular Atypia
15
6
15
4
0
2
11.32 ±2.37
12.84 ±8.73
10
11
10
9
0
2
11.00 ±2.12
12.53 ±6.45
13
8
13
6
0
2
10.44 ± 3.05
14.00 ± 6.48
(-)
(+)
Surgical Stage
I
II-IV
(")
Diploid Aneuploid
Mean ± SD
P
0.750
0.474
0.102
P = probability; SD = standard deviation; HPF = high-power fields.
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