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453
Loss of Expression of the Gene Deleted in Colon
Carcinoma (DCC) Is Closely Related to Histologic
Differentiation and Lymph Node Metastasis in
Endometrial Carcinoma
Makoto Saegusa, M.D.
Miki Hashimura, B.Ph.
Atsuko Hara, M.D.
Isao Okayasu, M.D.
Department of Pathology, Kitasato University
School of Medicine, Kanagawa, Japan.
BACKGROUND. Although frequent loss of the tumor suppressor gene deleted in
colon carcinoma (DCC) has been demonstrated in endometrial carcinoma, an
alteration of the expression during normal menstrual cycle and tumorigenesis from
hyperplastic lesions is still unclear.
METHODS. A total of 151 endometrial carcinomas (endometrioid type), along with
90 hyperplasias (23 simple, 30 complex, and 37 atypical) and 143 normal endometria (28 atrophic, 44 proliferative, and 71 secretory), were immunohistochemically
investigated for expression of DCC as well as for estrogen and progesterone
receptors (ER and PR). Analysis for DCC mRNA levels was also performed on 37
endometrial carcinomas and 14 normal endometria.
RESULTS. DCC expression was observed in endometrial glandular cells in both
proliferative and secretory stages; the immunoreactivity scores were not related to
values for either ER or PR. The values for DCC were significantly higher in hyperplasia than in normal endometria, and then decreased in the sequence leading to
Grade 3 carcinoma. In endometrial carcinoma, reduction or loss of DCC expression
was significantly related to the histologic evidence of malignancy and lymph node
metastasis, and this was in keeping with the results of mRNA analysis. The transcripts derived from alternative splicing in the extracellular domain were not
observed in any tumor samples.
CONCLUSIONS. The findings of this study indicate that DCC expression may be
linked to the maintenance of differentiated glandular cells during the normal
menstrual cycle without any relation to immunoreactivity for ovarian hormone
receptors. Moreover, loss or reduction of expression may be a significant event in
the progression of endometrial carcinoma through metastatic features. Cancer
1999;85:453– 64. © 1999 American Cancer Society.
KEYWORDS: endometrial carcinoma, endometrial hyperplasia, gene deleted in colon
carcinoma (DCC), estrogen receptor, progesterone receptor.
T
Address for reprints: Makoto Saegusa, M.D., Department of Pathology, Kitasato University School
of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan.
Received March 17, 1998; revision received June
22, 1998; accepted June 22, 1998.
© 1999 American Cancer Society
he gene DCC (deleted in colorectal carcinoma) has been identified
at chromosome 18q21, a common region of allelic loss in human
colorectal carcinomas.1 Genomic analysis has revealed that the open
reading frame of this gene consists of 29 exons spanning nearly 1.4
megabases, and that it encodes a 1447–amino acid transmembrane
protein with four immunoglobulinlike and six fibronectin type III–like
extracellular domains.2,3 DCC transcripts and protein are at low levels
in most tissues but are abundant in the central and peripheral nervous system, where they may play important roles in cell growth and
differentiation.3
454
CANCER January 15, 1999 / Volume 85 / Number 2
FIGURE 1. Immunoreactivity of the gene deleted in colon carcinoma (DCC) in normal and hyperplastic endometria is depicted. Semiserial sections of normal
endometrium are shown in proliferative (A,F) and secretory (B,G) stages and hyperplasias with complex (C,H), simple (D,I), and atypical (E,J) types. (A–E) H&E
staining; (F–J) DCC immunohistochemistry. (F,G) Moderate immunoreactivity for DCC is observed in endometrial glandular cells. High magnification (⫻200) of areas
indicated by arrows is illustrated in the insets. (H,I,J) Note the strong cytoplasmic binding in hyperplastic glandular components. (Original magnification ⫻100.)
The DCC gene has been considered as a candidate
tumor suppressor gene for a variety of human malignancies. Reduction of transcripts or allelic deletions of
this gene are frequently observed in prostate, breast,
esophageal, and brain tumors.4 –7 Moreover, it has
been demonstrated that full length, but not truncated,
DCC can suppress the tumorigenicity of a human
squamous carcinoma cell line.8
Endometrial carcinoma is a common malignancy
in the female reproductive tract, and its development
is closely related to change in ovarian hormone status.
Although frequent reduction or loss of DCC has also
been demonstrated in endometrial carcinomas,9,10
any association with estrogen and progesterone receptors is still unclear. In addition, to our knowledge
there has been no report regarding DCC expression
during the normal menstrual cycle and in hyperplastic
lesions.
In this study, to clarify the significance of DCC
gene abnormalities to the development and progression of endometrial carcinomas, we therefore investigated normal, hyperplastic, and malignant endometria, using immunohistochemistry and a combination
of the reverse transcriptase–polymerase chain reaction and the Southern blot hybridization (RT-PCR/
SBH) assays to determine expression levels. The results were compared with hormone receptor status
and a number of other prognostic factors, such as
clinical stage, degree of myometrial invasion, and
lymph node metastasis. In addition, it was examined
whether DCC inactivation is related to either loss of
heterozygosity or aberrant alternative splicing of
mRNA.
MATERIALS AND METHODS
Cases
A total of 151 cases of endometrial carcinoma (endometrioid-type), surgically resected at Kitasato University Hospital during the period 1988 –1997, were investigated. Ninety cases of hyperplasia (including 23
simple and 30 complex hyperplasias without atypia,
and 37 atypical hyperplasias), obtained by curettage or
hysterectomy, were also examined, along with 28 samples of atrophic (postmenopausal) endometrium adjacent to carcinoma samples and 115 normal biopsy
DCC Expression in Endometrial Carcinoma/Saegusa et al.
455
FIGURE 2. Immunoreactivity scores for the gene deleted in colon carcinoma (DCC), estrogen receptor (ER), and progesterone receptor (PR) are given for normal
(A) and hyperplastic (B) endometria. AE: atrophic endometrium; Pro: proliferative phase; Sec: secretory phase; SH: simple hyperplasia; CH: complex hyperplasia;
AH: atypical hyperplasia. The data are mean ⫾ standard deviation values.
specimens (44 proliferative and 71 secretory). All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Histologic diagnoses were made according to the criteria of the
International Federation of Gynecology and Obstetrics
(1988).11 The carcinoma cases comprised 89 Grade (G)
1, 39 G2, and 23 G3 lesions. Of all carcinoma cases
investigated, 143 tumors were available for examination of the correlation between immunohistochemical
parameters and clinicopathologic data, including clinical stage, degree of myometrial invasion, and lymph
node status. Areas of squamous differentiation within
tumors, including squamous metaplasia (Sq-M) and
morules, were classified after Zaino et al.12 Of 151
carcinomas, 20 cases with Sq-M foci and 16 tumors
with morule lesions were also investigated for whether
DCC immunoreactivity was altered in Sq-D areas. In
addition, 37 endometrioid carcinomas and 14 normal
endometrial samples were further snap-frozen in liquid nitrogen for RNA analysis.
Immunohistochemistry
Immunohistochemistry was performed using a combination of the microwave oven heating and the standard streptavidin-biotin-peroxidase complex (LSAB
kit, Dako, Copenhagen, Denmark) methods. Immunohistochemical detection of DCC protein was per-
formed as described by Shibata et al.13 Briefly, slides
were heated in 10 mM citrate buffer (pH 6.0) for six
5-minute cycles for DCC and three 5-minute cycles for
estrogen and progesterone receptors (ER and PR), using a microwave oven, and were then incubated overnight at 4°C with optimum dilutions of primary antibodies. The antibodies used were antihuman DCC
monoclonal antibody (clone G97-449, ⫻100 dilution,
Pharmingen, San Diego, CA), anti-ER mouse monoclonal antibody (⫻80 dilution, Novocastra Lab. Ltd.,
Newcastle, UK), and anti-PR mouse monoclonal antibody (⫻80 dilution, Novocastra Lab. Ltd., Newcastle,
UK). To confirm the specificity of binding, normal
mouse serum (⫻500 dilution) was supplied instead of
primary antibody as a negative control.
Scoring for DCC and Immunoreactivity of Hormone
Receptors
Scoring of the immunohistochemistry results was
done as previously reported.14 Briefly, based on the
percentages of immunopositive epithelial cells
among the totals of normal or neoplastic cells on a
slide section, there were 5 categories of subdivision,
as follows: 0, all negative; 1, ⬍10% positive cells; 2,
10 –30%; 3, 30 –50%; and 4, ⬎50%. The immunointensity was also subclassified into 4 groups, as follows: 0, negative; 1⫹, weak; 2⫹, moderate; and 3⫹,
456
CANCER January 15, 1999 / Volume 85 / Number 2
FIGURE 3. Immunoreactivity of the gene deleted in colon carcinoma (DCC) is depicted in endometrial carcinomas. (A–C) Semiserial sections of Grade (G) 1 (A,D),
G2 (B,E), and G3 (C,F) endometrioid carcinomas are shown. (A–C) H&E staining. (D–F) Note the strong (D), moderate (E), and negative (F) immunoreactivity. (Original
magnification ⫻200.)
strong. Immunoreactivity scores were generated by
multiplication of the values for the two parameters.
The scoring was performed by one of us (M.S.), the
evaluation being carried out as triplicate examinations.
Reverse Transcriptase–Polymerase Chain Reaction
Total cellular RNA was extracted from 37 tumors and
14 normal endometrial frozen tissues using Isogen
(Nippon Gene Co., Tokyo, Japan). cDNA was synthesized from 5 ␮g of total RNA using RAV-2 reverse
transcriptase (Takara, Shiga, Japan) in the presence of
random primers (Takara) and a ribonuclease inhibitor
(Takara) in a 20 ␮L reaction volume at 42°C for 60
minutes. One ␮L of cDNA solution was amplified by
Taq polymerase (Takara) in a volume of 10 ␮L. For
detection of DCC mRNA expression in exons 6 –7,
primers used were 5⬘-TTCCGCCATGGTTTTTAAATCA-3⬘ for the sense and 5⬘-AGCCTCATTTTCAGCCACACA-3⬘ for the antisense, published by Fearon et
al.1 For analysis of alternative splicing in the extracellular domain (exon 17), primers used were DCK2834S,
5⬘-CCCAGACTAACTGCATCATCATGAG-3⬘ (sense),
and DCK3151A, 5⬘-CACCTACTGGTGGGAGCAT-3⬘
(antisense), described by Reale et al.15 The PCR procedure was performed with 35 cycles of denaturation
at 94°C for 0.5 minutes, annealing at 55°C for 1 minute,
and extension at 72°C for 1 minute, with a final extension time of 7 minutes. As a negative control, water
was supplied instead of template DNA for each examination. To examine the quality and quantity of the
synthesized cDNA, the ␤-actin specific primers (sense,
5⬘TGCTATCCAGGCTGTGCTAT-3⬘ and antisense, 5⬘GATGGAGTTGAAGGTAGTTT-3⬘) were also ampli-
DCC Expression in Endometrial Carcinoma/Saegusa et al.
457
FIGURE 4. (A) Immunoreactivity scores for the gene deleted in colon carcinoma (DCC), estrogen receptor (ER), and progesterone receptor (PR) are given for normal
and hyperplastic endometria and endometrial carcinomas. N: normal endometrium, including atrophic, proliferative, and secretory stages; H: hyperplasia; G: grade.
The data are mean ⫾ standard deviation (SD) values. (B) Immunoreactivity scores for primary carcinomas (Ca) and areas of squamous differentiation, including
squamous metaplasia (SqM) and morules (M), are shown. The data are mean ⫾ SD values.
fied.16 PCR assays were performed in duplicate or
triplicate.
Southern Blot Hybridization
A 10 ␮L aliquot of each PCR reaction mixture was
electrophoresed in a 3.0% agarose gel and transferred
to a Hybond N nylon membrane (Amersham, Tokyo,
Japan). After prehybridization using DIG Easy Hyb
solution (Boehringer Mannheim, Tokyo, Japan), filters
were hybridized overnight with each digoxigenin-labeled exon-specific probe, which corresponded to internal sites between primer sets used. The sequences
of oligonucleotide probes for DCC exon 6 –7, DCC
exon 17, and ␤-actin were as follows: probe DCC exon
6/7 (5⬘-AATTGGATGAAGAATGGAGATGTGGTCATT3⬘, encoding nucleotides 1087–1116 in the cDNA sequence), probe DCC exon 17 (5⬘-ATGAGTTGGACTCCTCCCTTGAAC-3⬘, nucleotides 2226 –2250), and
probe ␤-actin (5⬘-ACTGACTACCTCATGAAGATCCTCACCGAG-3⬘, nucleotides 597– 626). Hybridization
signals were detected with a DIG Luminescent Detection Kit (Boehringer Mannheim). The conditions used
for hybridization, washing, and detection were in
keeping with the manufacturer’s recommendations.
Between each hybridization, the filter was stripped
before being rehybridized with another probe.
RT-PCR/SBH Data Analysis
Quantitation of hybridization signals for DCC exon
6/7 and ␤-actin, respectively, was performed by densitometric analysis using NIH Image version 1.58 software. The relative expression level of DCC mRNA was
calculated by normalization to the hybridization signals for ␤-actin in each case as follows: value for DCC
mRNA signal, divided by that for ␤-actin. Based on the
average values for relative DCC transcripts in normal
endometria, subdivision was into 4 categories, as follows: negative, ⬍10% of normal; ⫾, 10 –50%; 1⫹, 50 –
100%; 2⫹, ⬎100%, according to the methods described by Enomoto et al., with minor modification.9 A
458
CANCER January 15, 1999 / Volume 85 / Number 2
FIGURE 5. The relation between gene deleted in colon carcinoma (DCC) immunoreactivity scores and clinicopathologic factors for endometrial carcinomas is
shown. U: upper half; L: lower half; P: positive; N: negative. The data are mean ⫾ standard deviation values.
loss or reduction of DCC expression was concluded
with scores of either negative or ⫾, and a positive one
with 1⫹ or 2⫹.
Detection of alternative splicing within DCC exon
17 was performed as described by Reale et al.15 Briefly,
DCK2834S and DCK3151A primers generated a product of 341 bp if the more 5⬘ splice acceptor at exon 17
was used (60-nucleotide sequence present) or an alternatively spliced product of 281 bp if the more 3⬘
splice acceptor site was used.
Detection of Loss of Heterozygosity (LOH) by PCR Assay
Genomic DNAs were extracted using GenomicPrepTM
cells and tissue DNA isolation kit (Pharmacia Biotech,
Tokyo, Japan). Aliquots of 100 ng DNA were used as
templates in a reaction volume of 10 ␮L. For detection
of LOH at the DCC locus, a PCR-LOH assay was performed using a primer set recognizing a variable number of tandem repeats (VNTR) within the DCC gene.
The sequences of primers were 5⬘-GATGACATTTTCCCTCTAG-3⬘ (sense) and 5⬘-GTGGTTATTGCCTTGAAAAG-3⬘ (antisense),6 and the PCR procedure was
carried out with 32 cycles of 0.5 minutes at 94°C, 1
minute at 57°C, and 1 minute at 72°C, along with a
predenature step of 2 minutes at 94°C and postextension for 5 minutes at 72°C. The PCR products were
electrophoresed on 3% agarose gels and then stained
by ethidium bromide. LOH was defined as a more
than a 50% decrease in relative intensity of alleles on
comparison of amplified alleles between normal and
tumor DNAs, as described previously.17
Statistics
Statistical analysis of data for immunoreactivity scores
was performed using the Mann–Whitney U test and
the Pearson’s correlation coefficient. Association of
RT-PCR data for DCC transcripts between exon 6/7
and 17 regions were examined by the chi-square test.
The cutoff for statistical significance was defined as
P ⬍ 0.05.
RESULTS
DCC Expression in Normal and Hyperplastic Endometria
Weak-to-moderate cytoplasmic immunoreactivity for
DCC was found in the endometrial glandular cells.
The positivity and intensity appeared to be similar in
DCC Expression in Endometrial Carcinoma/Saegusa et al.
459
FIGURE 6. Serial sections of morule (A,C)
and squamous metaplastic (B,D) areas are
shown. (A,B) H&E staining. Immunoreactivity
of the gene deleted in colon carcinoma
(DCC) is strong in a morule (C) and weak in
a squamous metaplastic focus (D). (Original
magnification ⫻200.)
both proliferative and secretory phases. In some cases,
stromal cells also exhibited weak binding, whereas
other endometrial components, including myometrium and vessels, lacked immunoreactivity (Fig.
1A,B,F,G). In atrophic (postmenopausal) endometria,
DCC positive glandular cells were observed with relatively weak immunointensity.
Moderate-to-strong immunoreactivity for DCC
was observed in simple or complex hyperplastic lesions, the positive cells being distributed homogeneously with cytoplasmic staining. The immunostaining pattern of atypical hyperplasia was the same. Weak
immunoreactivity in stromal cells was also noted in
some cases (Fig. 1C,D,E,H,I,J).
Antibodies against ER and PR diffusely reacted
with several endometrial components, including glandular, stromal, and myometrial cells, with distinct nuclear staining.
No alteration of average values for DCC immuno-
reactivity scores were observed during normal menstrual cycles, whereas marked reduction was noted for
both ER and PR with transition from the proliferative
to the secretory phase. The average immunoreactivity
scores for atrophic endometria were significantly
lower than in either proliferative or secretory values
(Fig. 2A). In addition, the average values for DCC were
not significantly different among simple, complex,
and atypical hyperplasias (Fig. 2B).
DCC Expression in Endometrial Carcinomas
A variety of immunostaining patterns for DCC were
observed in endometrial carcinomas. In some cases,
DCC positive tumor cells were diffusely distributed
with strong cytoplasmic staining and some membrane
binding, whereas completely lacking or sporadic
weak-to-moderate positivity were observed in other
tumors (Fig. 3).
460
CANCER January 15, 1999 / Volume 85 / Number 2
TABLE 1
Correlation of Immunoreactivity Scores among DCC, ER, and PR
Em Ca
G1
G2
G3
Hyperplasia
Simple
Complex
Atypical
Normal epithelium
Atrophic
P-phase
S-phase
n
DCC vs. ER
r (P value)
DCC vs. PR
r (P value)
ER vs. PR r (P
value)
89
39
23
0.32 (0.002)
0.39 (0.015)
0.42 (0.05)
0.13 (0.2)
0.33 (0.04)
0.28 (0.2)
0.41 (⬍0.0001)
0.13 (0.45)
0.39 (0.06)
23
30
37
0.06 (0.79)
0.27 (0.16)
0.02 (0.89)
0.02 (0.92)
0.24 (0.21)
0.03 (0.85)
0.17 (0.4)
0.72 (⬍0.0001)
0.19 (0.26)
28
44
71
0.08 (0.69)
0.004 (0.98)
0.26 (0.03)
0.44 (0.02)
0.26 (0.09)
0.09 (0.45)
0.08 (0.69)
0.32 (0.03)
0.86 (⬍0.0001)
DCC: gene deleted in colon carcinoma; ER: estrogen receptor; PR: progesterone receptor; n: no. of cases; r: Pearson’s correlation coefficient; Em Ca: endometrial
carcinoma; G: grade; P-phase: proliferative phase; S-phase: secretory phase.
The average values for DCC scores decreased in
the sequence leading from hyperplasias to Grade 3
carcinomas, the difference being significant, as well as
ER and PR status, whereas the scores in hyperplastic
lesions were significantly higher than in normal endometrium at any stage (Fig. 4A).
As shown in Figure 5, the DCC scores in the negative lymph node metastasis group were significantly
higher than in the positive category, but there was no
correlation between the scores and either the clinical
stage or the degree of myometrial invasion.
The DCC antibody also bound to areas of squamous differentiation within tumors, showing strong
immunoreactivity in morules and weak or negative
staining in squamous metaplastic (Sq-M) lesions (Fig.
6). Average immunoreactivity scores in morules were
significantly higher than in carcinomatous areas, in
contrast to the Sq-M lesions (Fig. 4B).
No correlation was evident between values for
DCC and either ER or PR in normal, hyperplastic, and
malignant endometrial lesions (Table 1).
transcripts and the immunoreactivity scores was observed for endometrial carcinomas (Fig. 8). As shown
in Table 2, reduction (⫾) or loss (⫺) of DCC expression
was observed in 13 (35.1%) of 37 carcinomas, when
subdivision was into 4 categories on the basis of the
average values for normal endometria.
With primer sets for alternative splicing sites
within DCC exon 17, only a 341 bp and not a 281 bp
fragment was amplified in 26 (70.3%) of 37 carcinomas (Fig. 7 and Table 2), the positivity being significantly related with that for DCC exon 6/7 transcripts (Table 3).
Loss of Heterozygosity of DCC
To determine whether alteration of DCC mRNA expression is related to DNA abnormalities, the PCRLOH assay was performed. With a primer set for the
VNTR region in the DCC locus, although 11 of 31
(35.5%) endometrial carcinomas were informative, no
LOH was identified (Table 2).
DISCUSSION
RT-PCR/SBH Assay
Detection of mRNAs for DCC and ␤-actin genes was
performed using a combination of RT-PCR and SBH
assays. RNAs obtained from 14 normal endometria
and 37 endometrial carcinomas could all be successfully amplified by ␤-actin specific primer sets, the
products showing the expected molecular weight of
446 bp (Fig. 7).
With primer sets corresponding to DCC exons 6 to
7, amplicons were observed with a molecular weight
of 233 bp (Fig. 7). Positive correlation (r ⫽ 0.567, P ⫽
0.0007) between the relative amounts of DCC exon 6/7
The current study immunohistochemically demonstrated that DCC expression is maintained from proliferative though to secretory stages totally, the immunoreactivity scores being not statistically related to the
ovarian hormone receptor status. Hedrich et al.3 proposed that, rather than physically holding cells together, DCC may be important in transmitting signals
from the cell’s exterior environment to its interior,
thereby playing a role in controlling differentiation.
Our findings therefore suggest that DCC may play
some role in the differentiative pathway of endometrial glandular cells through proliferative to secretory
DCC Expression in Endometrial Carcinoma/Saegusa et al.
461
FIGURE 7. A reverse transcriptase–
polymerase chain reaction/Southern blot
hybridization assay, for the gene deleted
in colon carcinoma (DCC) and ␤-actin in
endometrial carcinomas, is depicted.
FIGURE 8.
The correlation between
gene deleted in colon carcinoma (DCC)
immunoreactivity scores and relative
amounts of DCC mRNA in endometrial
carcinomas is shown.
phases without any relation to the expression of ovarian hormone receptors. The relative down-regulation
of DCC expression observed for atrophic endometrium may be related to the associated reduction in
their proliferative activity.
Kurman et al.18 asserted that the majority of hyperplasias, including simple, complex, and atypical
types, regress or remain stable rather than progressing. In this study, DCC expression was increased
rather than decreased in hyperplastic lesions com-
pared with normal endometrium, independent of the
histologic subtype. With regard to colorectal lesions,
DCC expression appears to be similarly increased in
hyperplastic polyps, with retention in early and intermediate stage adenomas but loss in some late adenomas with significant dysplasia.3 Goi et al.19 also reported that normal colonic and adenoma tissues
significantly expressed the DCC protein, in contrast to
colonic carcinomas showing poor expression. Our results indicate that loss of DCC expression is rare event
462
CANCER January 15, 1999 / Volume 85 / Number 2
TABLE 2
Data on the Relation between DCC mRNA Expression and Immunoreactivity in Endometrial Carcinomas
DCC exon 6/7
Case
no.
Grade
C1
G1
C2
G2
C3
G2
C4
G1
C5
G1
C6
G1
C7
G3
C8
G1
C9
G1
C10
G1
C11
G1
C12
G2
C13
G1
C14
G1
C15
G3
C16
G2
C17
G1
C18
G1
C19
G1
C20
G1
C21
G2
C22
G1
C23
G1
C24
G2
C25
G2
C27
G1
C29
G1
C31
G2
C32
G2
C35
G2
C36
G1
C38
G2
C39
G2
C40
G3
C41
G1
C42
G1
C43
G1
Normal (n ⫽ 14)
DCC exon 17
Stage
MI
LN
Ratio
mRNA*
281 bp
341 bp
IHC score
for DCC
DCC LOH
(VNTR)
I
I
III
I
I
I
I
II
I
I
I
I
I
IV
II
I
III
II
II
I
I
I
I
I
III
III
II
II
II
II
II
III
II
I
I
I
I
U
L
L
L
U
L
U
L
L
U
U
L
L
U
L
L
U
U
U
U
U
U
U
U
L
L
L
L
U
L
L
U
U
L
U
L
L
N
N
P
N
N
N
N
P
N
N
N
N
N
P
N
N
N
N
N
N
N
N
N
N
N
P
N
N
N
N
NE
NE
N
P
N
N
P
0.95
0.01
0.54
0.72
0.98
0.9
0.9
1.4
0.96
0.45
0.12
0.51
0.48
0.93
0.86
0.97
1.15
0.23
0
0.16
0.7
0.36
1.6
1.31
1.12
0.27
1.15
0.31
1.29
0.95
0
0.41
0.72
0.28
1.56
1.48
0.75
0.97 ⫾ 0.38
1⫹
⫺
1⫹
1⫹
2⫹
1⫹
1⫹
2⫹
1⫹
⫾
⫾
1⫹
⫾
1⫹
1⫹
1⫹
2⫹
⫾
⫺
⫾
1⫹
⫾
2⫹
2⫹
2⫹
⫾
2⫹
⫾
2⫹
1⫹
⫺
⫾
⫹
⫾
2⫹
2⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
⫺
⫹
⫺
⫹
⫹
⫹
⫹
ND
4
ND
4
6
8
8
12
12
9
4
3
4
6
8
9
4
4
2
4
2
2
ND
1
4
4
8
4
8
6
2
ND
ND
2
8
8
6
6.0 ⫾ 3.1
N
H
H
H
H
H
H
N
N
H
H
H
N
N
N
H
H
H
H
N
N
H
H
H
H
N
H
N
H
N
H
ND
ND
ND
ND
ND
ND
DCC: deleted in colon carcinoma gene; MI: degree of tumor myometrial invasion; U: upper half of the myometrium; L: lower half of the myometrium; LN: lymph node status; N: negative; P: positive; NE: not examined;
*: relative abundance of DCC mRNA; (⫺: ⬍10% of average value for normal endometrium; ⫾: 10–50%; 1⫹: 50–100%; 2⫹: ⬎100%); IHC: immunohistochemistry; ND: not done; LOH: loss of heterozygosity; VNTR:
variable number of tandem repeats; N: negative; H: homozygous (not informative).
in hyperplasias, suggesting that up-regulation of the
expression in these lesions may be associated with
accelerated proliferation and differentiative features.
Frequent reduction or loss of DCC expression has
been documented in a variety of human malignancies,
such as colorectal, pancreatic, and prostatic adenocarcinomas.4,20,21 In the colorectal case, DCC expression
is markedly reduced during tumor progression from
intramucosal to invasive tumors with loss detected in
100% of tumors with liver metastasis, suggesting that
DCC inactivation is a late event in tumor progression
and that the molecule may act as a suppressor of
metastasis.21,22 In tumors of the female reproductive
tract, Enomoto et al.9 demonstrated impaired DCC
expression in 4 (50%) of 8 endometrial and 12 (55%) of
22 ovarian epithelial tumors, particularly tumors with
higher histologic grade, suggesting an association with
an aggressive phenotype. Gima et al.10 described loss
of DCC expression in 14 (50%) of 28 endometrial carcinomas but did not correlate with histologic differentiation and progression (clinical stage). In our large
series, reduction or loss of DCC protein was signifi-
DCC Expression in Endometrial Carcinoma/Saegusa et al.
463
TABLE 3
Relation between Exon 6/7 and Exon 17 DCC mRNA Expression in Endometrial Carcinomas
DCC exon 17 expression
DCC exon 6/7
expression
Positive
Negative
Positive
Negative
P value
21 (56.8)
5 (13.5)
3 (8.1)
8 (21.6)
0.0019
DCC: gene deleted in colon carcinoma.
cantly related to the histologic malignancy and lymph
node metastasis in endometrial carcinomas, in line
with the results of mRNA assay. Considering no association with FIGO stage or degree of myometrial invasion, the alteration of expression may be closely linked
with the tumor progression through metastatic potential. Further studies in this area are clearly warranted.
In most human malignancies, inactivation of a
tumor suppressor gene commonly appears to be due
to a point mutation of one allele and loss of the remaining allele. For DCC inactivation, aberrant splicing
and allele-specific loss of transcripts have also been
proposed to be important mechanisms.15,23 Reale et
al.15 have identified two alternatively spliced regions
in the DCC gene, involving extracellular (exon 17) and
cytoplasmic (exon 26) domain sequences. In our
study, however, no alternative splicing transcripts in
extracellular domain sequences could be found in endometrial carcinomas. Although we did not examine
alternative splicing in cytoplasmic domain sequences,
the results thus suggest that in endometrial carcinomas, alternative splicing of DCC transcripts may be
less important than inactivation at the gene level.
Loss of heterozygosity (LOH) of the DCC gene has
been demonstrated in tumors of the female reproductive tract, for example, in 1 (13%) of 8 and 4 (31%) of 13
endometrial carcinomas by Okamoto et al.24 and
Imamura et al.,25 respectively, using the probe OLVII
E10. LOH of chromosome 18q detected by the three
probes, pHH64, OLVII E10 and OS-4, was also reported for 16 (26%) of 61 endometrial carcinomas.10
However, in the current study using the PCR-LOH
assay, no LOH on the VNTR region of the DCC locus
was found, even in carcinomas demonstrating loss of
mRNA expression. The possible reasons for anomalous results include the following: 1) inevitable contamination of noncancerous cells in the tumor samples may mask any genetic deletion; 2) other regions
of the DCC gene may be affected by the deletion or
rearrangement. Recently, Kong et al.26 demonstrated
that in neuroblastomas LOH is not necessary for DCC
gene inactivation and that other mechanisms, such as
mutations in the coding regions or alterations in initiator or promoter areas, can contribute. Similar findings have also been reported for osteosarcomas and
endometrial carcinomas.9,16
Another interesting finding in the current study
was the alteration in DCC expression in morules and
squamous metaplastic (Sq-M) lesions within tumors.
We previously demonstrated that the morule foci may
be intermediate in phenotype between primary tumors and Sq-M lesions, using bcl-2 immunohistochemistry,27 but the current findings suggest a more
complex scenario. Clearly, further studies of the relation between DCC expression and cell differentiation
are warranted.
In conclusion, the current study demonstrated
that DCC expression is maintained during the normal
menstrual cycle without any relation to expression of
ovarian hormone receptors. In addition, loss or reduction of expression may be a specific event in the progression of endometrial carcinomas through metastatic features.
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