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h t . J. Cancer (Pred. Oncol,): 69,170-173 (1996)
0 1996 Wiley-Liss, Inc.
(*
&- - .
Publication of the International Union Against Cancer
Publication de I'UnionInternationale Contre le Cancer
IMMUNOHISTOCHEMICAL DETECTION OF ADHESION MOLECULE
CD44 SPLICE VARIANTS IN LYMPH NODE METASTASES
OF CERVICAL CANCER
Christian KAINZ~.~,
Clemens TEMPFER',
Petra KOHLBERGER',
Seline JANISCHI,
Heinz KOELBL',Gerald GITSCH'
and Gerhard BREITENECKER~
'Departntent of Gynecology and Obstetricsand ?Instituteof Pathology, Gynecopathological Unit,
University of Vienna Medical School, Kenna, Austria.
Expression of specific cell adhesion molecule CD44 isoforms
(splice variants) has been shown to be associated with poor
prognosis in human cervical cancer. We used 3 different variant
exon sequence-specific murine monoclonal antibodies (MAbs)
to epitopes encoded by exons v5, v6 and v7-v8 of human variant
CD44 to study the expression of CD44 splice variants in 35
primary squamous-cell carcinomas of the cervix and pelvic
lymph node metastases by means of immunohistochemistry.
Primary tumors showed expression of CD44 splice variants
CD44v5, CD44v6 and CD44v7-8 in 93%, 73% and 33% of cases,
respectively. Lymph node metastases expressed CD44v5,
CD44v6 and CD44v7-8 in 83%, 53% and 2 I YOof cases, respectively. Tumors with expression of CD44v6 in pelvic lymph node
metastases showed metastaticspread to 2 or more pelvic lymph
nodes significantly more often compared to patients without
expression of splice variant CD44v6. Patients suffering from
tumors with lymph node metastases expressing splice variant
CD44v6 had a poorer recurrence-free survival compared to
patients without CD44v6 expression in lymph node metastases,
but this trend was not statistically significant. Expression of
CD44 splice variants containing epitopes encoded by exon v6 in
primary tumors and pelvic lymph node metastases of cervical
cancer patients is consistent with a prominent role of CD44 in
the process of metastasisformation.
o 1996 Wiley-Liss,Inc.
The transmembrane receptor protein CD44 belongs to the
family of cell surface adhesion molecules involved in cell-cell
and cell-matrix interactions (Jalkanen et al., 1987). CD44
mediates lymphocytic functions such as cell activation, motility, division, adhesion to the extracellular matrix and adhesion
to stromal cells (Huet et al., 1989; Stamenkovic et al., 1991).
CD44 expression is postulated to control lymphocytic recirculation by mediating lymphocytic binding to peripheral lymph
nodes and mucosal and synovial endothelial cells (Jalkanen et
al., 1987). Various human malignancies express isoforms of
CD44, characterized by the insertion of additional amino acids
in the extracellular domain of the CD44 protein (Gunthert et
a/., 1991; Screaton et al., 1992). CD44 proteins are encoded by
a gene located on chromosome 11. By post-translational
modifications of pre-messenger RNA, i.e., alternative splicing,
numerous isoforms of the CD44 protein are produced (CD44
splice variants CD44vl-CD44v10) (Screaton et a/., 1992; Smith
et a/., 1989; Matsumura and Tarin, 1992). Aberrant expression
of CD44 splice variants, eg., CD44 splice variants V6 and
V7-8. in human tumors indicates a loss of splice control in
malignantly transformed cells (Heider et al., 1993) and has
been shown to be associated with metastasis and poor prognosis in breast cancer, colorectal cancer and gastro-intestinal
lymphoma (Kaufmann et a/., 1995; Wielenga et a/., 1993;
Joensuu et a/., 1993). In previous studies, we showed that in
cervical cancer over-expression of CD44v6 is associated with
poor prognosis (Kainz et a/., 1995~).In cervical cancer stage
111, over-expression of CD44v6 and CD44v7-8 is correlated
with unfavorable prognosis (Kainz et al., 1995b).
Considering the physiological function of CD44, it has been
speculated that malignant cells expressing CD44 splice variants are mimicking lymphocytes and thus manage to perform
the cell-cell and cell-matrix interactions necessary for metastatic spread (Dall et al., 1994; Herrlich et al., 1993~).An
analogous expression pattern of CD44 variants in primary
tumors and regional lymph nodes would support the hypothesis that aberrant expression of CD44 adhesion molecules on
tumor cells is a crucial condition of their capability to form
metastases. Another point of interest is whether expression of
CD44 variants can be used to identify patients with a better
prognosis within the high-risk collective of lymph nodepositive patients. To address these questions we examined the
expression of CD44 splice variants CD44v5, CD44v6 and
CD44v7-8 in primary tumor and lymph node samples of 35
patients with surgically treated cervical cancer stages IB to IIB.
PATIENTS AND METHODS
Patients
Thirty-five patients with surgically treated squamous-cell
carcinoma (SCC) of the cervix and metastatic disease in the
pelvic lymph nodes were included in the study. In all women
diagnosis was established pre-operatively by biopsy or conisation. All patients were treated by radical hysterectomy and
pelvic lymphadenectomy between 1984 and 1990 and underwent post-operative adjuvant radiotherapy.
Staging was done according to UICC criteria. Stages IB, IIA
and IIB were present in 14, 5 and 16 cases, respectively.
Histologically, tumors were graded as well (Gl, n = l l ) ,
moderately (G2, n = 16) and poorly (G3, n = 8) differentiated. All patients underwent a close follow-up scheme consisting of regular visits at 3-month intervals, including clinical
examination with vagino-rectal palpation and serology with
tumor marker evaluation. Ultrasound examination of the
pelvis and chest X-ray were performed every 6 months. In
cases of suspect clinical findings or when serology showed
elevated marker levels, computed tomography was performed.
The follow-up period ranged between 3 and 8 years (median
length 51.7 months).
From each lymph node specimen three different slides with
tumor cells present were chosen for staining. From the primary
tumor specimens, 1 slide representing the bulk of the primary
tumor was chosen for staining. For CD44 splice variant
analysis, we used 3 different variant exon sequence-specific
murine monoclonal antibodies (MAbs) to epitopes encoded by
exons v5, v6 and v7-v8 of human variant CD44.
lmmunohistochemistly
We prepared all sections from routinely formalin-fixed and
paraffin-embedded surgery samples to evaluate the expression
whom correspondence and reprint requests should be sent, at
Department of Gynecology and Obstetrics, Vienna University Medical
School, A-1090 Vienna, Spitalg. 23, Austria. Fax: (43) 1-40400-2907.
Received: November 8,1995 and in revised form January 22,1996.
CD44 IN CERVICAL CANCER
of epitopes encoded by CD44 splice variants CD44v5, CD44v6
and CD44v7-8. Sections were deparaffinized in xylene and
rehydrated in a graded alcohol series (100% to 70%). To
recover antigenicity, we used the Antigen Retrieval System
(BioGenex, San Ramon, CA), twice for 20 min in a microwave
at 600 W power (HM 146, Elektra Bregenz, Schwaz, Austria),
then rinsed sections in 10 mM PBS (pH 7.6). Three different
variant exon sequence-specific murine MAbs were used: the
first was a MAb specific for the epitope encoded by exon v5 of
human variant CD44 (CD44v5, clone VFF-8; Bender, Vienna,
Austria), the second MAb was specific for the epitope encoded
by exon v6 of human variant CD44 (CD44v6, clone VFF-7;
Bender) and the third MAb was specific for the epitope
encoded by exons v7-8 of human variant CD44 (CD44v7-8,
clone VFF-17; Bender). The primary antibody was diluted in
serum/PBS and sections incubated for 60 min and then for a
further 30 min with biotinylated anti-mouse and anti-rabbit
link antibody (LSAB 2 Kit, DAKO, Carpinteria, CA). After
rinsing in PBS, sections were coated with streptavidin conjugated to alkaline phosphatase for 10 min. Sections were then
rinsed in PBS, incubated with Fast red chromogen (naphtol
phosphate substrate in Tris buffer, Fast red chromogen tablets
5 mg; BioGenex) and then washed with distilled water.
Sections were finally counter-stained with hematoxylin and
mounted. Staining was judged as positive when ( i ) more than
20% of the tumor area showed strong membrane staining or
(ii) more than 80% of the tumor area showed weak but
unequivocal membrane staining. All other cases were judged
as being negative.
Positive control. The positive control slide was prepared from
epidermal tissue, known to contain the antigen. In the positive
control tissue, all MAbs were stained similarly.
Negative control. The negative control slide was prepared
from the same tissue block as the specimen. Instead of the
primary antibody we used a normal, non-immune serum
supernatant from the same source as the primary antibody.
Statistics
The x2 test was used where appropriate. Results were
analyzed for the end-point of overall and recurrence-free
survival. Overall survival was defined as the period between
the date of primary surgery and death. Survival times of
patients still alive were censored with the last follow-up date
(last follow-up date or date of death), respectively. Survival
probabilities were calculated by the product limit method of
Kaplan and Meier (1958). Differences between groups were
tested using the Wilcoxon test. All p values are results of
2-sided tests. The BMDP statistical software system (BMDP
Statistical Software, Los Angeles, CA) was used for calculations.
171
FIGURE
1- Expression of CD44-containing epitope v6 in invasive cervical carcinoma tissue (white arrows); cervical stroma cells
are negative. Carcinoma in situ sections are seen as well (black
arrows). Bar = 40 km, counter-stain, hematoxylin.
FIGURE
2 - Expression of CD44-containingepito e v6 in metastatic tumor cells in a pelvic lymph node specimen. 8D44v6 is seen
on the cell membrane of tumor cells. Bar = 20 km, counter-stain,
hematoxylin.
shows expression of CD44v6 on the cell membranes of a
cervical carcinoma specimen. We examined only invasive
carcinomas. However, areas of carcinoma in situ were present
in some areas, as shown in Figure 1. In these cases, there was
no difference in CD44 expression between the invasive section
of the tumor and the carcinoma in situ (Fig. 1). Figure 2 shows
expression of CD44v6 on the cell membranes of metastatic
RESULTS
tumor cells in a pelvic lymph node specimen. The expression.
patterns of CD44 splice variants in primary tumors and
Median age of the patients was 43.4 (SD 11.6, minimum 26, corresponding lymph node metastases are shown in Table I.
maximum 71) years. Mean number of nodes removed during
Tumors with expression of CD44v6 in pelvic lymph node
pelvic lymphadenectomy was 16.2 (8 minimum, 35 maximum).
metastases
showed metastatic spread to 2 or more pelvic lymph
Primary tumors showed expression of CD44 splice variants
CD44v5, CD44v6 and CD44v7-8 in 93%, 73% and 33% of nodes significantly more often compared with patients without
cases, respectively. There was no correlation of CD44 expres- expression of splice variant CD44v6 (59% vs. 23%, x2 test,
sion to tumor stage or grading. Lymph node metastases p = 0.05). Expression of splice variants CD44v5 and CD44v7-8
expressed CD44v5, CD44v6 and CD44v7-8 in 83%, 53% and in pelvic lymph node metastasis showed no correlation with the
21% of cases, respectively. We found homogenous staining in extent of lymph node metastasis.
Expression of the 3 examined CD44 splice variants in the
tumors and lymph nodes that were considered positive for
CD44 expression. In all positive cases, we found a membrane primary tumor was of no prognostic value. Patients suffering
staining pattern. No cytoplasmic staining was present. Figure 1 from tumors with lymph node metastases expressing splice
KAINZ ETAL.
172
TABLE I - EXPRESSION PATTERN OF CDJ4 SPLICE VARIANTS CD44v5,
CD44v6 AND CD44v7-8 IN PRIMARY TUMORS AND CORRESPONDING
MFTASTATIC LYMPH NODES
~
Tumor-uositive, metastasispositive
Tumor-positive, metastasisnegative
Tumor-negative, metastasisnegative
Tumor-negative, metastasispositive
1
.=:
1.0
3
8
CD44y5
expression
CD44y6
expression
CD4Jvl-8
expresston
+
83%
53%
21%
C
10%
20%
12%
7%
277%
67%
0%
0%
0%
e
0
variant CD44v6 had a poorer recurrence-free survival compared with patients without CD44v6 expression in lymph node
metastases (Wilcoxon test, p = 0.07), but this trend was
not statistically significant. Kaplan-Meier analysis regarding recurrence-free survival of patients with and without
CD44v6 expression in pelvic lymph node metastases is shown
in Figure 3. Overall survival was not significantly associated
with CD44v5, CD44v6 or CD44v7-8 expression in lymph node
metastases (Wilcoxon test, p = 0.1, p = 0.09, p = 0.4, respectively).
E9
n
0.5
I
I
I
'1I
No CD44 v6Ixpression
CD44 v6-Expression
Q
g
0
20
40
60
80
100 120 140
Months Since Initial Treatment
FIGURE3 - Overall survival of patients suffering from tumors
with or without expression of splice variant CD44v6 in pelvic
lymph node metastases (Wilcoxon test,p = 0.07).
DISCUSSION
We investigated 35 patients with cervical cancer and metastatic spread to the regional lymph nodes to learn more about
the role of the cell adhesion molecule CD44 in tumor metastasis.
Others have hypothesized that expression of the cell adhesion molecule CD44 plays a crucial role in the natural history
of the metastatic process (Dall et al., 1994; Stamenkovic et al.,
1991). In a previous study, we supported this hypothesis by
finding a significant correlation between expression of CD44
splice variants and the probability of metastasis in cervical
cancer patients (Kainz et al., 199%). Expression of CD44 in
lymph node metastasis of cervical cancer, however, has not
been investigated. We found that CD44 splice variants are
expressed in metastatic tumor cells of cervical cancer patients.
Given the hypothesis that a cell clone with high expression of
CD44v6 would possess a selection advantage in the process of
metastasis formation (Herrlich et al., 1993a, b ) , we expected a
higher rate of CD44v6 expression in metastatic lymph nodes
compared with primary tumor. However, we found a lower
expreTsion rate in the metastatic lymph nodes for all 3 splice
variants of CD44 investigated (Table I). A possible explanation for CD44 down-regulation could be found in the influence
of a different organic environment altering the expression
pattern of cell surface molecules in regional lymph nodes.
Shedding of membrane-bound molecules after having accomplished their function could also account for a weaker staining
in regional lymph nodes compared with primary tumor (Dolo
et al., 1994; Shibata et al., 1993).
In a previous study, we found a prognostic value for CD44v6
expression in the primary tumor of patients free of pelvic
lymph node metastasis. Contrary to that finding, expression of
splice variants CD44v5, CD44v6 and CD44v7-8 in primary
tumor failed to predict a poor outcome in patients displaying
metastatic disease in pelvic lymph nodes (Kainz et al., 1995~).
Such a high-risk collective, consisting only of patients with
histologically proven metastatic spread to the regional lymph
nodes, was further investigdted in the present study.
As expected, expression of CD44v6 in primary tumor was
not predictive of outcome. We could show that expression of
CD44v6 in the metastatic tumor cells of pelvic lymph nodes is
associated with a trend to poorer prognosis (Fig. 1). If our
results are confirmed in a larger series of patients, expression
of CD44v6 in pelvic lymph node metastasis could be used as a
means to identify patients with a favorable prognosis within
the high-risk collective of node-positive patients.
In a previous study, we also showed that the incidence of
pelvic lymph node metastasis is significantly higher when
primary tumors express CD44v6 (Kainz et a/., 1 9 9 5 ~ )In
. the
present study, a significantly higher number of positive lymph
nodes was found when CD44v6 expression was seen in metastatic lymph nodes. These results underline again the functional role of CD44v6-expressing cells in the expansion of
cervical cancer beyond regional limits.
Although the expression rate of CD44 splice variants in
metastatic lymph nodes was lower than in primary tumor, we
found that CD44v6 expression in pelvic lymph node metastases
is associated with a trend to poorer prognosis and that it is
predictive of the extent of pelvic lymph node metastasis. We
conclude that tumors which conserve their capability of CD44v6
expression in the altered environment of pelvic lymph nodes
have the worst prognosis.
We showed that CD44 splice variants are expressed in pelvic
lymph node metastasis. Expression of CD44v6 in lymph node
metastases of cervical cancer patients is a promising marker
for identifying patients with a favorable prognosis within the
high-risk collective of node-positive patients. The functional
role of CD44 splice variant expression in the natural history of
metastasis is underlined by the correlation between CD44v6
expression and the extent of pelvic lymph node involvement in
cervical cancer patients.
ACKNOWLEDGEMENTS
This work was supported by a research grant from the mayor
of Vienna (1045) to C.K.
CD44 IN CERVICAL CANCER
173
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