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1691
Lack of CD44 Variant 6 Expression in Advanced
Extrahepatic Bile Duct/Ampullary Carcinoma
Yujiro Yokoyama, M.D.1
Eiso Hiyama, M.D.2
Yoshiaki Murakami, M.D.1
Yuichiro Matsuura, M.D.1
Takashi Yokoyama, M.D.2
1
First Department of Surgery, Hiroshima University Faculty of Medicine, School of Medicine, Hiroshima, Japan.
2
Department of General Medicine, Hiroshima University Faculty of Medicine, School of Medicine,
Hiroshima, Japan.
BACKGROUND. Transmembrane proteins of the CD44 family play roles in cell-cell
and cell-matrix interactions, and their aberrant expression has been reported to be
associated with the growth and metastasis of various tumors. The authors examined CD44 standard (CD44st) and CD44 variant 6 (CD44v6) expression in extrahepatic bile duct (EHBD)/ampullary carcinoma.
METHODS. In 36 EHBD/ampullary carcinomas, immunohistochemical analyses
with monoclonal antibodies against the human CD44st protein or CD44v6 protein
were performed by the streptavidin-biotin immunoperoxidase method. The relation between expression of the proteins and lymph node metastases or patient
outcome was investigated. To verify the lack of CD44v6 mRNA expression in
tumors negative for CD44v6 staining, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization were performed.
RESULTS. Immunohistochemical analyses revealed that 13 of 36 carcinomas
(36.1%) expressed the CD44v6 protein. Only 2 of 13 CD44v6-expressing primary
tumors (15.4%) had regional lymph node metastases, whereas 14 of 23 tumors
showing no CD44v6 expression (60.9%) had lymph node metastases (Fisher exact
test, P , 0.01). Moreover, a lack of CD44v6 expression was correlated significantly
with poor prognosis (generalized Wilcoxon test, P , 0.05). Eleven of 13 CD44v6
positive tumors showed CD44st expression, which also was correlated with prognosis. RT-PCR and Southern blot hybridization revealed a lack of CD44v6 mRNA
expression in the tumors that were negative for CD44v6 staining.
CONCLUSIONS. The results of the current study suggest that a lack of expression of
CD44, especially CD44v6, is correlated with lymph node metastases and poor
prognosis, and may be a prognostic factor for patients with EHBD/ampullary
carcinoma. Cancer 1999;86:1691–9. © 1999 American Cancer Society.
KEYWORDS: CD44, extrahepatic bile duct, ampulla of Vater, cancer, lymph node
metastases, prognosis.
Presented at the 16th World Congress Collegium
Internationale Chirurgiae Digestive, Madrid, Spain,
September 16 –19, 1998.
The authors thank Dr. T. Kodama for his helpful
suggestion and Ms. Fukuba for her excellent technical assistance.
Address for reprints: Yujiro Yokoyama, M.D., First
Department of Surgery, Hiroshima University Faculty of Medicine School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
Received December 31, 1998; revision received
April 29, 1999; accepted June 15, 1999.
© 1999 American Cancer Society
T
umor metastases and invasion are the major causes of death in
patients with cancer. Because the extrahepatic bile duct (EHBD)
has no thick muscular layer, bile duct carcinoma can disseminate
easily. Moreover, invasion to large vessels, such as the portal vein or
the hepatic artery, occurs frequently, making surgical extirpation
difficult.
Recently, cell adhesion molecules, such as CD44, E-cadherin, KAI1
(CD82), and integrin, have been reported to play major roles in invasion
and metastases of a variety of epithelial and nonepithelial malignant
cells. In gastric carcinoma, a decrease of E-cadherin expression was
reported to be correlated with poor prognosis of the patients.1 KAI1
protein expression decreased during the progression of prostatic carcinoma.2 Decreased expression of the integrin a-5 subunit was reported to
1692
CANCER November 1, 1999 / Volume 86 / Number 9
be correlated with more malignant phenotypes of human hepatocellular carcinoma.3
CD44, which is one of the well-known cell adhesion molecules, is a polymorphic transmembrane
glycoprotein whose gene is located at chromosome
11p and comprises 20 exons that span 50 kilobases.4
CD44 molecules are expressed as several isoforms
generated by alternative splicing of exons of the
CD44 gene. Recently, correlations between clinicopathologic features of various cancers and CD44
expression have been reported. In solid malignant
tumors, high levels of expression of CD44 protein
have been reported in advanced breast carcinoma,5
gastric carcinoma,6 multiple myeloma,7 colorectal
carcinoma,8 –10 and vulval carcinoma.11 Conversely,
CD44 expression has been reported to decrease in
high grade and poorly differentiated types of bladder carcinoma,12 prostate carcinoma,13 ovarian carcinoma,14 neuroblastoma,15,16 endometrial carcinoma,17 and oral squamous cell carcinoma.18 Thus, the
correlation between CD44 expression and tumor
malignancy appears to differ in different types of
tumors. Because few detailed studies of CD44 expression in EHBD/ampullary carcinoma have been
reported, we analyzed the expression of CD44 variant 6 (CD44v6) and CD44 standard (CD44st) in these
malignancies and examined the correlation between
the expression of these molecules and the clinicopathologic profiles of the patients.
MATERIALS AND METHODS
Patients
Thirty-six patients with carcinoma of the EHBD/ampulla who underwent surgical resection in the First
Department of Surgery, Hiroshima University Hospital, were enrolled in this study. Patient characteristics
are shown in Table 1.
Clinicopathologic Features
There were 28 male patients and 8 female patients,
and their mean age at surgery was 63.6 years (range,
41– 83 years). Pathologic classification and clinical
staging were performed according to the criteria of the
International Union Against Cancer (UICC).19 Among
the 36 patients with EHBD/ampullary carcinomas, 25
patients had EHBD carcinoma (8 patients with Grade
1, 16 with Grade 2, and 1 with Grade 3 histopathology),
and 11 patients had ampullary carcinoma (6 patients
with Grade 1, 4 with Grade 2, and 1 with Grade 3
histopathology). There 6 patients had pT1 tumors, 21
patients had pT2 tumors, 8 patients had pT3 tumors,
and 1 patient had a pT4 tumor. Twenty patients had
no lymph node metastases, and 16 patients had more
than one lymph node metastasis. Six patients had
Stage I disease, 11 patients had Stage II disease, 14
patients had Stage III disease, and 5 patients Stage IV
disease.
Twelve patients died, and 13 were alive and disease free of the patients with EHBD carcinoma. Three
patients died, and 8 patients were alive and disease
free of the patients with ampullary carcinoma. The
mean follow-up time for these patients was 51.7
months (range, 0.8 –143.8 months) after surgical resection.
Immunohistochemistry
Tissues were fixed in 10% buffered formalin at room
temperature and embedded in paraffin for histologic
diagnosis. From these tissues, serial sections were cut,
mounted on gelatin-dichromate-coated glass slides,
deparaffinized, rehydrated, and pretreated with 3%
hydrogen peroxide for 15 minutes at room temperature to quench endogenous peroxidase activity. Then,
the sections were incubated with 10% normal rabbit
serum for 1 hour at room temperature and incubated
with the antihuman CD44v6 monoclonal antibody
(2F10; R&D Systems, Minneapolis, MN) and antiCD44st monoclonal antibody (F10-44-2; DAKO;
Carpinteria, CA) at 4 °C overnight in a moist chamber.
After washing in phosphate-buffered saline (PBS), the
sections were incubated with biotinylated rabbit antimouse immunoglobulin G (IgG), IgM, and IgA
(Nichirei, Tokyo, Japan) for 1 hour at room temperature, followed by rinsing in PBS, and then incubated
with peroxidase-conjugated streptavidin (Nichirei).
Positive sites were developed with 200 mg/mL of 3,39diaminobenzidine containing 0.04% hydrogen peroxide in PBS. Finally, the sections were counterstained
with Mayer hematoxylin and mounted. Negative control sections, which were incubated as described
above but without the primary antibody, showed no
staining. Samples that showed strong plasma membrane-type staining pattern were interpreted as positive, and samples that showed either no staining or
only cytoplasmic staining were interpreted as negative.13
RNA Isolation
Total RNA was extracted from two noncancerous
EHBD epithelium specimens and two EHBD carcinoma specimens, using the acid-guanidine-phenolchloroform (AGPC) method protocol.20 The purity and
concentration of RNA were determined with a spectrophotometer by calculating the A260/A280, ratio and
RNA samples were electrophoresed on 1.5% agarose
gels to confirm the presence of high-quality 18s and
28s bands.
CD44v6 in Bile Duct-Ampullary Carcinoma/Yokoyama et al.
1693
TABLE 1
Patient Features and Immunohistochemical Detection of CD44
Age
(yrs)/gender
Location
Prognosis
(months)/status
Grade
pTNMa
Stage
CD44v6
CD44st
53/M
60/F
50/M
41/M
57/M
71/M
62/M
72/M
83/M
72/M
58/M
70/Mb
60/Mc
60/F
73/F
67/M
64/F
61/M
58/M
67/F
62/M
63/F
68/M
64/M
77/M
69/M
57/F
72/F
59/M
62/M
66/M
70/M
52/M
71/M
67/M
50/M
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
EHBD
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
Va. Pap.
143/A
143/A
123/A
121/A
106/A
4/D
86/A
61/A
53/A
14/D
12/D
12/D
8/D
13/D
18/D
12/D
91/A
1/D
21/D
50/A
52/A
45/A
28/D
42/A
32/D
11/D
136/A
114/A
10/D
77/A
58/A
52/A
49/A
17/A
8/D
41/A
2
1
2
1
1
2
1
2
1
1
1
2
2
2
2
2
2
2
2
2
1
3
2
2
2
3
1
1
2
1
2
1
2
1
2
1
pT2pN0pM0
pT2pN0pM0
pT1bpN0pM0
pT3pN0pM0
pT2pN0pM0
pT3pN2pM0
pT1apN0pM0
pT2pN0pM0
pT2pN0pM0
pT3pN0pM0
pT3pN2pM0
pT2pN2pM0
pT2pN2pM0
pT2pN2pM0
pT2pN2pM0
pT2pN1pM0
pT2pN2pM0
pT2pN2pM0
pT2pN0pM0
pT2pN2pM0
pT2pN0pM0
pT2pN1pM0
pT2pN0pM0
pT2pN0pM0
pT2pN0pM0
pT3pN0pM0
pT1pN0pM0
pT2pN1pM0
pT3pN1pM0
pT2pN0pM0
pT3pN1pM0
pT1pN0pM0
pT4pN0pM0
pT1pN0pM0
pT3pN1pM0
pT1pN0pM0
II
III
I
IV-A
II
IV-A
I
II
II
IV-A
IV-A
III
III
III
III
III
III
III
II
III
II
III
II
II
II
II
I
III
III
II
III
I
IV
I
III
I
N
N
N
N
P
N
P
P
P
P
N
N
N
N
N
N
P
N
N
N
N
N
N
P
N
N
P
P
N
N
N
P
P
P
N
P
N
N
N
N
P
N
P
N
P
P
N
N
N
N
N
N
P
N
N
N
N
N
N
P
N
N
P
N
N
N
N
P
N
P
N
P
M: male; F: female; EHBD: extrahepatic bile duct; Va. Pap: Vater papilla; A: alive; D: dead; N: negative; P: positive.
a
Tumor classification is according to the TMN classification system (Hermanek and Sobin19).
b
Case 12.
c
Case 13 (shown in Fig. 2).
Reverse Transcriptase and Polymerase Chain Reaction
The first strand of cDNA was synthesized from purified total RNA using Superscript II™ (Gibco BRL Life
Technologies, Grand Island, NY). The procedure followed the manufacturer’s protocol, which can be
summarized as follows: The first-strand cDNA synthesis reaction from 1.0 mg of total RNA was catalyzed by
RNase H- reverse transcriptase (RT) using oligo (dT) to
hybridize to 3-poly (A) tails. The cDNA was amplified
for 30 cycles of polymerase chain reaction (PCR) under
the following conditions: 94 °C for 1 minute, 55 °C for
1 minute, and 72 °C for 2 minutes. The sequences of
the two primers, P1 and P2, in this PCR were as follows21: primer P1: 59-CAGACCTGCCCAATGCCTTTGATGGACC; primer P2: 59-CAAAGCCAAGGCCAAGAGGGATGCC. These primers were against a 445base pair (bp) portion of the basic CD44 standard
gene. The PCR was carried out in a final volume of 50
mL containing the following: 1.0 mL cDNA; 1.5 mM
MgCl2, 0.25 mM each of dNTP; 25 mM each of primer
(P1 and P2); 25 mM ammonium sulfate buffer, pH 9.5;
and 2.5 U Taq polymerase (TaKaRa, Kyoto, Japan).
Five microliters of the PCR product were electrophoresed in a 2% agarose gel and stained with ethidium
1694
CANCER November 1, 1999 / Volume 86 / Number 9
bromide. RNA from each sample was processed in
parallel without the using the reverse transcriptase
(RT) as a control to determine that the product obtained was of cDNA and not a result of contamination
by genomic DNA. Moreover, mRNA of a housekeeping
gene, glyceraldehyde-3-phosphate dehydrogenase
(G3PDH), was amplified by RT-PCR in each case as a
marker of the integrity of the extracted RNA.
Southern Blot Hybridization
Twenty microliters of each PCR product were electrophoresed in 1% agarose gels and subjected to Southern blot transfer onto nitrocellulose filters. CD44 exon
11 (v6) specific probe was PCR amplified from
genomic DNA derived from normal human peripheral
blood cells with the following primers22: primer P3: 59
ACTCAGGCAACTCCTAGTAGTACAACG-39 primer P4:
59-TGTCCCTGTTGTCGAATGGGAGTCTTC-39 The amplified fragment (125 bp) was labeled by a-32P-dTP
using the random primer method and then hybridized
with the filter. After washing three times, the filter was
exposed for 12 hours to Fuji X-ray film (Fuji, Tokyo,
Japan) at room temperature.
Statistical Analysis
Comparisons between CD44v6 expression in tumor
tissues and clinicopathologic parameters were performed using the chi-squared test or the Fisher exact
test, as appropriate. The Kaplan–Meier survival curves
were used to determine survival rates,23 and the differences in survival rates between subgroups of patients were compared using the generalized Wilcoxon
test.
RESULTS
Immunohistochemistry
CD44v6 expression was examined in 29 noncancerous
EHBD samples but not in the remaining 7 samples,
which had lost noncancerous EHBD epithelium due to
chronic cholangitis. Two of 29 samples (6.9%), which
were degenerated severely and detached from the bile
duct wall, showed no immunoreactivity. The remaining 27 noncancerous epithelial tissue samples (93.1%)
showed strong plasma membrane-type staining patterns. A similar expression pattern was observed in 13
tumors but not in the remaining 23 tumor samples
(63.9%). Figure 1 shows noncancerous EHBD epithelium (Fig. 1A) and a cancerous specimen (Fig. 1B)
from the same patient.
Noncancerous EHBD samples showed positive
staining patterns for CD44st similar to those for
CD44v6. All CD44v6 negative samples also were negative for staining by CD44st. Except for 3 samples, all
CD44v6 positive samples were stained positively for
CD44st.
RT-PCR and Southern Blot Hybridization
To analyze the mRNA transcription levels in the tissues without expression of CD44v6 as detected by
immunostaining, RT-PCR and Southern blot hybridization were performed as follows: The cDNA from the
2 cancerous tissue samples and corresponding noncancerous EHBD epithelium samples were examined
to verify the lack of CD44v6 mRNA expression in the
CD44v6 negative tumors. The cDNA from 2 noncancerous EHBD epithelium samples yielded prominent,
standard-sized CD44 PCR products. The amplified
products in these 4 samples varied in size between 445
bp and 1450 bp. Prior to CD44 specific PCR amplification, a housekeeping gene (G3PDH) was amplified
successfully using cDNA derived from the purified
total RNA in all 4 tissue samples, indicating the integrity of the extracted RNA (data not shown). Negative
controls for each of these 4 samples using the cellular
RNA were processed in parallel without the RT enzyme to rule out the possibility of genomic DNA contamination.
Hybridization with a CD44v6 specific probe
yielded a complex pattern of bands in the two noncancerous EHBD epithelium specimens analyzed.
However, CD44v6 mRNA was not detected in the 2
EHBD carcinoma specimens (Fig. 2).
CD44v6 Status and Clinicopathologic Parameters
The correlation between CD44v6 expression in
EHBD/ampullary carcinoma and clinicopathologic
findings is shown in Table 2. In histopathologic
grading, only 4 CD44v6 positive tumors (30.8%)
were Grade 2 or Grade 3; conversely, 18 CD44v6
negative tumors (78.3%) were Grade 2 or Grade 3
(P , 0.05). Among the EHBD carcinoma samples, 14
of 18 CD44v6 negative tumors (77.8%) were Grade 2
or Grade 3, and 4 of 7 CD44v6 positive tumors
(57.1%) were Grade 1. Among ampullary carcinoma
samples, 4 of 5 CD44v6 negative tumors (80.0%) and
only 1 CD44v6 positive tumor were Grade 2 or Grade
3, but not significantly. Only 2 of 13 CD44v6 positive
tumors (15.4%) had invaded regional lymph nodes,
whereas 14 of 23 CD44v6 negative tumors (60.9%)
had lymph node metastases (P , 0.01). Among the
EHBD carcinoma samples, 10 of 18 CD44v6 negative
tumors had invaded regional lymph nodes, whereas
6 of 7 CD44v6 positive tumors lacked lymph node
metastases. Among the ampullary carcinoma samples, only 1 CD44v6 positive tumors showed lymph
node metastases. Eleven of 13 CD44v6 positive tumors (84.6%) were classified as Stage I or II, whereas
CD44v6 in Bile Duct-Ampullary Carcinoma/Yokoyama et al.
1695
FIGURE 1. (A) Noncancerous extrahepatic bile duct epithelium with diffuse,
strong, membranous reactivity for CD44
variant 6 (CD44v6) (antihuman CD44v6,
clone 2F10 with Mayer hematoxylin
counterstain; original magnification,
3200). (B) Tumor cells in extrahepatic
bile duct carcinoma showing the absence of CD44v6 protein expression (antihuman CD44v6, clone 2F10 with
Mayer hematoxylin counterstain; original
magnification, 3200).
14 of 23 CD44v6 negative tumors (60.9%) were classified as Stage III or IV, but the differences between
CD44v6 positive tumors and CD44v6 negative tumors were not significant.
To evaluate the difference between early tumors,
the correlation between CD44v6 expression and clinicopathologic findings was investigated in patients
with pT1 or pT2 tumors (Table 3). There was a significant correlation between CD44v6 reactivity and histologic grade, and CD44v6 negative carcinoma, especially CD44v6 negative EHBD carcinoma, tended to
have more frequent lymph node metastases, but the
increase was not significant.
CD44v6 Status and Patient Prognosis
Figure 3A shows survival curves for the patients with
CD44v6 positive ampullary carcinoma and those with
CD44v6 negative carcinoma. In these 2 groups, the
5-year overall survival rates were 100% and 40.0%,
respectively. Figure 3B shows survival curves for the
patients with CD44v6 positive EHBD carcinoma and
1696
CANCER November 1, 1999 / Volume 86 / Number 9
TABLE 2
Correlation between Expression of CD44v6 and Clinicopathologic
Parameters in 36 Patients with Extrahepatic Bile Duct and Ampulla
Carcinoma
Parameter
Histologic grade (1:2 or 3)b
EHBD
Va. Pap
Primary tumor (pT1 or 2:pT3 or 4)b
EHBD
Va. Pap
Regional lymph node metastases
(negative:positive)
EHBD
Va. Pap
Stage grouping (I or II:III or IV)b
EHBD
Va. Pap
CD44v6
negative
CD44v6
positive
P valuea
5:18
4:14
1:4
16:7
15:3
1:4
9:4
4:3
5:1
11:2
6:1
5:1
,0.05
N.S.
N.S.
N.S.
N.S.
N.S.
9:14
8:10
1:4
8:15
6:12
2:3
11:2
6:1
5:1
9:4
5:2
4:2
,0.01
N.S.
N.S.
N.S.
N.S.
N.S.
EHBD: extrahepatic bile duct; Va. Pap: Vater papilla; N.S.: not significant.
a
Fisher exact test.
b
Histopathologic grading, primary tumor, stage grouping: Tumor classification is according to the
TMN classification system (Hermanek and Sobin19).
TABLE 3
Correlation between Expression of CD44v6 and Clinicopathologic
Parameters in 27 Patients with pT1 or pT2 Carcinoma in
Extrahepatic Bile Duct and Ampulla
Parameter
FIGURE 2.
Reverse transcriptase-polymerase chain reaction (RT-PCR) and
Southern blot hybridization. CD44 PCR products were hybridized with a CD44
variant 6 (CD44v6) specific probe. The products derived from both noncancerous extrahepatic bile duct epithelium samples, but not those from immunohistochemically CD44v6 negative tumors, were hybridized with CD44v6 specific probes. No.: case number (shown in Table 1); T: extrahepatic bile
duct/ampullary carcinoma; N: noncancerous extrahepatic bile duct epithelium;
bp: base pairs.
those with CD44v6 negative carcinoma. In these 2
groups, the 5-year overall survival rates were 85.7%
and 38.9%, respectively. The prognosis of patients
with CD44v6 negative carcinoma was significantly
poorer compared with that of patients with CD44v6
positive carcinoma (generalized Wilcoxon test: P ,
0.05). No patients with CD44v6 positive ampullary
carcinoma died. Three of 5 patients with CD44v6 negative ampullary carcinoma died: 2 from peritoneal
carcinomatoses and 1 from liver metastases. Only 1 of
the 7 patients with CD44v6 positive EHBD carcinoma
died of liver metastases, whereas 11 of 18 patients with
CD44v6 negative EHBD carcinoma died: 4 from peritoneal carcinomatoses, 3 from liver metastases, and 4
Histologic grade (1:2 or 3)b
EHBD
Va. Pap
Regional lymph node metastases
(negative:positive)
EHBD
Va. Pap
Stage grouping (I or II:III or IV)b
EHBD
Va. Pap
CD44v6
negative
CD44v6
positive
P valuea
3:13
2:13
1:0
8:3
3:3
5:0
,0.05
N.S.
N.S.
7:9
7:8
0:1
7:9
6:9
1:0
9:2
5:1
4:1
9:2
5:1
4:1
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
EHBD: extrahepatic bile duct; Va. Pap: Vater papilla; N.S.: not significant.
a
Fisher exact test.
b
Histopathologic grading, stage grouping: Tumor classification is according to the TMN classification
system (Hermanek and Sobin19).
from disease progression the details of which were
unknown.
All patients with pT1 or pT2 ampullary carcinoma
are alive disease free. Figure 3C shows survival curves
for the 2 groups of patients with pT1 or pT2 EHBD
carcinoma. In these 2 groups, the 5-year overall survival rates were 100.0% and 40.0%, respectively.
Among the patients with pT1 or pT2 EHBD carcinoma,
CD44v6 positive carcinoma also was correlated significantly with favorable prognosis (P , 0.05).
CD44v6 in Bile Duct-Ampullary Carcinoma/Yokoyama et al.
1697
FIGURE 3. (A) Overall survival curves of patients suffering from ampullary
carcinoma with or without CD44 variant 6 (CD44v6) expression. The survival
rate of patients with CD44v6 positive tumors was significantly higher compared
with that of patients with CD44v6 negative tumors (P , 0.05). (B) Overall
survival curves of patients with extrahepatic bile duct carcinoma with or
without CD44v6 expression. The survival rate of patients with CD44v6 positive
tumors was significantly higher compared with that of patients with CD44v6
negative tumors (P , 0.05). (C) Survival curves of patients with pT1 or pT2
extrahepatic bile duct carcinoma with or without CD44v6 expression. The
survival rate of the patients with CD44v6 positive tumors also was significantly
higher compared with that of patients with CD44v6 negative tumors (P , 0.05).
CD44st Expression
The correlations between CD44st expression and clinicopathologic features were similar to those for
CD44v6. The 5-year survival rates of patients with
CD44st positive and CD44st negative tumors were
90.0% and 46.2%, respectively, and the difference also
was significant (P , 0.05).
DISCUSSION
Lymph node metastasis is one of the factors that is
considered to affect surgical outcome in patients with
EHBD/ampullary carcinoma.24 –27 Although many imaging techniques, such as ultrasonography, computed
tomography, and magnetic resonance imaging, have
been performed to detect lymph node metastases,
they are difficult to detect in patients with these tumors before surgery. Therefore, other techniques for
predicting the existence of lymph node metastases
and patient prognosis would be very useful in the
determination of therapeutic regimens for improving
patient outcome.
Some studies have reported that the lack of certain CD44 isoforms is linked to tumor progression and
metastases in patients with bladder carcinoma,12
prostate carcinoma,13 ovarian carcinoma,14 neuroblastoma,15,16 endometrial carcinoma,17 and oral
squamous cell carcinoma.18
Fujita et al.17 reported that patients with CD44
negative endometrial carcinoma had a high incidence
of lymph-vascular space involvement. They postulated that the decrease in CD44 expression in endometrial carcinoma cells may cause reduced adhesion
between cells as well between cells and the basement
membrane, resulting in easier detachment from the
tissue.
We believe that, in patients with EHBD/ampullary
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CANCER November 1, 1999 / Volume 86 / Number 9
carcinoma, the lack of CD44 expression may lead to
decreased cell-cell adhesion, changes in the cells’ arrangement, and thus to detachment of the cells from
the basement membrane, enabling cancer cell migration to both local and distant sites, causing increased
regional lymph node metastases.
Ashida et al.28 reported that CD44st expression
was correlated significantly with the absence of metastases in 47 patients with cholangiocarcinoma. Our
study also indicated that the prognosis of patients
with CD44st positive tumors tended to be more favorable compared with that of patients with CD44st negative tumors, but the difference was not significant.
In the current study, almost all noncancerous
EHBD epithelium expressed CD44v6 epitope, as
shown by immunohistochemical analysis. We therefore consider that normal bile duct epithelium expresses CD44v6 and epitopes. EHBD epithelial cells
stained positively for CD44v6 and in the cell membranes adjacent to the basement membrane and
neighboring cells. Therefore, CD44 molecules are supposed to mediate adherence of cells to the basement
membrane and to other cells in EHBD epithelium.
Conversely, 63.9% of EHBD/ampullary carcinoma
samples did not express CD44v6 epitope (Table 1),
which suggests that some cancer cells had lost their
CD44v6 immunoreactivity during carcinogenesis or
disease progression. CD44v6 negative tumors frequently were classified in more advanced stages,
mainly as a result of more frequent lymph node metastases, compared with CD44v6 positive tumors (Table 2). We believe that the high metastatic potential of
tumors with a lack of CD44v6 expression is likely to
cause the short survival period of the patients.
One difference in the clinical behavior of EHBD
carcinoma and ampullary carcinoma was seen as reported previously.29 In that report, the survival rate of
patients with ampullary carcinoma was higher than
for those with EHBD carcinoma. To understand the
course of progression of these tumors, it may be useful
to compare the CD44v6 positive and negative groups
at the same stage in the same disease. Figure 3C shows
that lack of CD44v6 immunoreactivity may be a predictive factor of poor prognosis for patients with early
(pT1 or pT2) EHBD carcinoma. The poor prognosis of
patients with ampullary carcinoma also correlated
with the lack of CD44v6 expression (P , 0.05). Because
all patients with pT1 or pT2 ampullary carcinoma have
survived regardless of CD44v6 immunoreactivity, the
lack of CD44v6 expression is considered to be a predictive factor of poor prognosis for patients with advanced (pT3 or pT4) ampullary carcinoma.
The fact that 6 of 15 deceased patients died of
peritoneal carcinomatoses indicates that recurrence
easily may occur from cells that detach from primary
lesions that lack CD44 or lymph node metastases. We
speculate that a lack of CD44v6 leads tumor cells to
break out of the normal cell alignment, which causes
a worse differentiation grade and easier detachment
from the primary lesion and causes tumor cells to
invade regional lymph nodes. Even if macroscopic
regional lymph adenectomy is performed completely
by surgery, tumor cells sometimes remain, and some
residual tumor cells disseminate to the peritoneal
space and cause peritoneal carcinomatoses, so that
the prognosis is poor for these patients.
We conclude that a lack of CD44v6 expression in
EHBD/ampullary carcinomas is correlated with lymph
node metastases and poor patient prognosis. Thus,
CD44v6 expression in tumor cells may be a useful
prognostic indicator. More aggressive therapy may be
necessary for treating patients with carcinoma that
lacks CD44v6 expression.
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