Annals of Oncology 11: 295-300, 2000.
© 2000 Kluwer Academic Publishers. Primed in the Netherlands.
Original article
Expression of CD44 variant isoforms and association to the benign
form of endocrine pancreatic tumours
H. Imam, B. Eriksson & K. Oberg
Department of Internal medicine. University Hospital, Uppsala, Sweden
Summary
Background: The expression of CD44 and its isoforms have
been shown in many neoplastic tissues to serve as prognostic
indicators, therefore, the feasibility of using these as prognostic
markers in endocrine pancreatic tumour patients was examined.
Patients and methods: Immunohistochemistry (IHC) was
performed on 26 tumour samples (5 gastrinomas, 3 glucagonomas, 10 non-functioning tumours, 6 insulinomas, 2 mixed
insulinoma and glucagonomas) with monoclonal antibodies
against CD44s (standard form) and variant isoforms (v4, v5,
v6, v7, v7-8, v9, vlO). Staining was correlated to the tumour
proliferation, malignancy, metastasis and patients survival.
Results: There was variable expression of CD44s. All tumours showed complex expression of many isoforms. CD44v6
and CD44v9 were down regulated in malignant tumours.
There was statistical significance of CD44v6 expression in
benign tumours (P < 0.05) compared to malignant tumours
Introduction
CD44, a large family of trans-membrane glycoproteins
thought to be involved in processes, such as, extracellular
matrix binding, hemopoiesis, lymphocyte homing, cell
migration, embryonic development and tumour metastasis [1-4]. These variety of functions are mediated
through different isoforms of CD44 surface proteins
which are generated by differential splicing of the CD44
precursor mRNA, resulting in the insertion of up to
10 different additional exons into the standard mRNA
[5]. Variants containing alternatively spliced exon v6
(CD44v6) have been demonstrated to be able to confer
metastatic potential to a rat pancreatic carcinoma cell
line [6]. The identification of CD44 variant sequences in
human tumour cell lines [7] accelerated the increasing
number of analysis of human tumour materials. The
result of these studies indicate potential involvement of
CD44 variant isoforms in human tumour dissemination
[8, 9].
Endocrine pancreatic tumours (EPT) are solitary or
multiple neuroendocrine neoplasms with ability to produce hormones [10]. These tumours are classified either
as functioning or non-functioning depending on clinical
syndrome related to its hormone production [11]. The
and near significance in CD44v9 expression (P - 0.0574).
Survival of the patients with CD44v6 positive staining was
higher than those who were negative (P = 0.0822). Moreover,
the expression was well correlated to the patients without any
distant metastases (CD44v6, p< 0.001; CD44v9, P < 0.01).
Tumour proliferation (Ki67 index) correlated directly to the
malignancy (P < 0.05) and there was inverse correlation between Ki67 index and CD44v6 (P < 0.05) as well as v9
(P < 0.05).
Conclusions: Endocrine pancreatic tumours express CD44s
and isoforms differentially. Expression of the two isoforms of
CD44, namely v6 and v9 seem to be related more to benign
form of the tumour and could serve as a predictor of good
prognosis.
Key words: CD44 isoforms, endocrine pancreatic tumour,
immunohistochemistry, tumour proliferation
growth pattern of these tumours is heterogeneous, ranging from benign insulin-producing tumours to highly
malignant tumours with rapid growth. Survival of these
patients is fairly high depending on slow growth as well
as early diagnosis and treatment. Prediction of biologic
aggressiveness by means of tissue analysis is a good
means to select particular treatment regimen like
chemotherapy or biotherapy suitable for long term treatment.
Several reports have shown that expression of different
CD44 isoforms could be used as prognostic factor, but
the results are not consistent with the tumour types.
Some tumours have positive relation, while others have
negative relation to the tumour progression [12-16].
Dall and co-workers [17] have compared the difference
between RT-PCR and IHC. They concluded that IHC is
the better method for screening though the increased
sensitivity of RT-PCR can help identifying those
tumours where only few cells are positive. In a previous
study, involvement of CD44 standard and variant isoforms were investigated by RT-PCR in EPTs [18]. We,
therefore, studied the expression of CD44s and variant
isoforms (v4, v5, v6, v7, v7-8, v9, and vlO) performing
IHC with monoclonal antibodies. The possibility of
using these in the clinic for screening purposes was
296
investigated by correlating the expression pattern of
CD44s and its isoforms with tumour proliferation determined by Ki-67 index and presence or absence of metastases.
Patients and methods
Statistical analysis
Chi-square test was performed to compare the expression of different
CD44 isoforms and Ki-67 in malignant and benign tumours. Chisquare test was also performed to compare between CD44 expression
and Ki-67 index as well as metastasis. Survival analysis was performed
with Kaplan-Meier method and log-rank test, /"-value <0.05 was
considered significant.
Tissue samples
Specimens were obtained either during operation or by ultra-sound
guided needle biopsy. A total of 26 patients (5 gastrinomas, 6 insulinomas, 3 glucagonomas, 10 non-functioning and 2-mixed insulinoma
and glucagonoma) were included in the study. The average age of the
patients was 55 years (range 21-86 years). Four of the insulinomas, one
gastrinoma and two non-functioning tumours were considered benign
due to lack of metastasis and the encapsulated nature of the tumours.
All others were malignant and had either distant metastasis or local
invasive growth. Sixteen of all the patients were untreated when the
tissue was obtained. IFN-oc was given to one patient, octreotide to
one patient and chemotherapy was given to six patients. Two patients were treated with a combination of IFN-a and chemotherapy.
Duration of disease was median 55 months (range 6-168 months)
(Table 1).
Results
Normal human pancreas
Acinar cells and ductal epithelium stained strongly
positive for antibody against CD44s while the normal
islet cells were not stained (Figure 1A). CD44v4 and v7
are not expressed in normal pancreas. Antibody against
CD44v7-8 stained acinar and ductal cells. CD44v5 is
not expressed in normal islets, but very few acinar and
ductular cells are positive for the antibody against it.
Acinar and ductal epithelium stained positive for v6
(Figure IB), v9 and vlO. These antibodies did not stain
islet cells.
Immunohlstochemistry
The ABC immunoperoxidase method was performed as described
before [19]. In brief, 6 um thick serial sections were cut onto
chrom-gelatine coated slides and fixed in 100% acetone for 10 min.
Endogenous peroxidase and avidin binding protein were blocked by
0.3% H2O2 and avidin-biotin blocking kit (blocking kit, Vector Laboratories, Burlingame, California), respectively. Horse serum diluted
1:5 in phosphate buffered saline (PBS) was employed to avoid unspecific antibody binding. The monoclonal Ki-67 antibody and monoclonal CD44 antibodies (Ki-67, Dianova, Hamburg, Germany, CD44s
- DAKO, Copenhagen, Denmark; CD44s - British Biotechnology;
CD44 v4, v5, v6, v7, v7-8, vlO - Bender MedSystems, Vienna, Austria
and CD44v9 - Seikagaku Co. Tokyo, Japan) were applied for 1.5 hours
at room temperature or over-night at 4°C. Immunoreaction was
visualised with an Elite kit (Vector Laboratories), 0.02% H2O2 as
substrate and 3-amino-9-ethylcarbazol (Sigma, St. Louis, Missouri)
dissolved in dimethylsulfoxide as chromogene. Later counter-stained
with Meyer's hematoxylin. To avoid day to day variation, immunostaining with one antibody on all the samples was performed in a
single run including positive and negative control
All samples were examined to identify and characterise the tumour.
Formalin-fixed and paraffin embedded sections were used for the
morphological studies, argyrophil (Grimehus) staining [20] as well as
immunostaining with an antibody against chromogranin-A [21].
Evaluation of the staining
Immunostained slides were examined under light microscope. Expression of CD44s and its isoforms were grouped as, (—), no staining; (+),
weak staining intensity; (++), moderately high staining intensity of at
least 50% of the tumour cells and (+++), strong staining of all tumour
cells. Stained sections were examined by two independent observer and
~ 15% of borderline cases with discordant results, a consensus score
was obtained by the two observers examining the samples together.
Tumour proliferation was measured as percentage of Ki-67 stained
cells in a certain viewfield(Ki67 index). It was calculated on the three
most concentrated areas of positive cells. Tumours having > 10%
positive cells were grouped as highly proliferating tumours, 5%-10%
positive cells as moderately high proliferating and < 5% positive cells
as low proliferating tumours.
Expression of CD44 and correlation to malignancy
The immunohistochemical staining was variable with
antibodies against different CD44 variant isoforms.
Staining with antibodies against CD44 v4, v7 and vlO
were present in the tumour cell cytoplasm. CD44s, v6
(Figure 1C), v7-8 and v9 (Figure ID) showed cytoplasmic and membrane staining, whereas v5 had nuclear
staining.
The results of the immunohistochemical staining are
summarised in Table 1. We obtained positive staining
with the antibody against CD44s in all 26 samples. In
three of the samples (one non-functioning, one insulinoma, one mixed insulinoma and glucagonoma) only
few cells were positive (Table 1). The isoform CD44vlO
was also expressed in all specimens. However, CD44v4
and v7 seemed to be of low abundance. The CD44v6
staining was positive in 10 tumours; 6 of 7 benign and 4
of 19 malignant. Three of the CD44v6 positive tissues
had moderate staining and 7 had weak staining (Table 1).
The CD44v6 staining correlated to a benign tumour
phenotype (P < 0.05). The CD44v9 immunostaining
was more frequently positive in benign tumours (6 of 7)
than in malignant ones (8 of 19) (P - 0.0574).
Ki-67 index
All the tumour samples tested were positively stained
with Ki-67 antibody. Ki-67 index was 11.51 ± 1.81 (mean
± SEM) in malignant tumours and 5.55 ± 1.26 in benign
tumours (P < 0.05). Ki-67 index was compared with
the expression of CD44 and its isoforms. CD44 v6 and
v9 were found to be inversely correlated (P < 0.05 in
both).
297
Table 1. Summary of immunohistochemical staining of EPTs with antibodies against CD44 standard and different variant isofonns.
Pi
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Diagnosis
Metastases
Treatment
Duration of CD44s CD44v4 CD44v5 CD44v6 CD44v7 CD44v7-8 CD44v9 CD44vlO Ki-67
disease
index
(months)
Gastnnoma (benign)
Gastrinoma
(malignant)
Gastrinoma
(malignant)
Gastnnoma
(malignant)
Gastnnoma
(malignant)
Nonfunctioning
(malignant)
Nonfunctioning
(malignant)
Nonfunctioning
(malignant)
Nonfunctioning
(malignant)
Nonfunclioning
(malignant)
Nonfunctioning
(malignant)
Nonfunctioning
(benign)
Nonfunctioning
(benign)
Nonfunctioning
(malignant)
Nonfunctioning
(malignant)
Insulinoma (benign)
Insulinoma (benign)
Insulinoma
(malignant)
Insulinoma (benign)
Insulinoma (benign)
Insulinoma
(malignant)
None
None
IFN
0
162
122
+++
++
+
0.76
6.34
106
++
-
4.35
Glucagonoma
(malignant)
Glucagonoma
(malignant)
Glucagonoma
(malignant)
Insulinoma +
glucagonoma
(malignant)
26 Insulinoma +
glucagonoma
(malignant)
25
Lymph node 0
Liver
Chem
26 #
++
-
22.79
Liver
Chem
46
++
+
4.35
34 #
+++
+
6.39
++
-
8.72
13#
++
+
9.79
Lymph node 0
63 #
+ few
cells
+
15.27
Lymph node IFN + chem
+ liver
0
Liver
99#
++
+
8.90
67 #
++
-
6.10
None
0
55
++
-
8.11
None
0
48
++
-
4 72
Liver
Chem
15#
++
+
30.90
None
0
38
++
-
18.77
None
None
Liver
0
0
0
109
51
52
++
++
++
+
_
2.04
8.90
13.79
None
None
Liver
0
0
40
6#
168
++
++
+
+
+
-
9 14
5.24"
6.15
6.08
Lymph node, 0
later liver
Lymph node 0
Liver
Chem
Chem
111
some
cells
87
++
+ few
cells
Lymph node 0
56
++
-
Lymph node 0
+ liver
111
++
Lymph node
SMS
12.11
7.29
++
some
cells
Liver
Chem
23 #
+++
_
8.86
Liver
IFN +chem
37 #
+ few
cells
-
18.14
Abbreviations: IFN - interferon-alpha, SMS - octreotide; Chem - chemotherapy, 0 - untreated.
Symbols used are: # indicates death, - no staining; + weak staining intensity; ++ moderately strong staining intensity of at least 50% of tumor cells; +++ all tumor
cells strongly stained.
* Considered as low proliferating
Correlation with metastasis
Survival
Expression of CD44 and its isoforms were compared The mean duration of clinical follow-up was 66 months
with the presence of metastasis of the patients. Seventeen (range 6-168 months). Of the seven benign EPTs one
of twenty-six patients had either liver or lymph node patient died after six months of diagnosis and it was due
metastases or metastases at both sites. Nine patients did to complication of the operation, rest of the patients
not have any such metastasis (Table 1). Down-regulation were still alive at the time of the last follow-up. Survival
of CD44v6 and v9 significantly correlated with the curve obtained by Kaplan-Meier method shows that
presence of metastasis (P < 0.001 and P < 0.01, respec- CD44v6 positive patients had better survival than negative patients (Figure 2) did (P - 0.0822).
tively).
298
Figure I. (A) Immunohistochemical staining of a normal human pancreas, stained by CD44s antibody, showing staining to the acinar and
ductular cell, but islet cells are negative (x 100). (B) Normal pancreas stained with CD44v6 (x 100). (C) Benign insulinoma showing positive cell
membrane and cytoplasmic staining with an antibody against CD44v6 (x 200) (D) Same benign insulinoma showing positive staining with an
antibody against CD44v9 (x 200).
Discussion
The adhesion molecule CD44 and its isoforms have
been investigated in different tumour types. Expression
of different CD44 isoforms have been suggested to contribute to metastatic conversion or spread of different
human carcinomas [22-24]. The expression pattern and
relation to either survival or malignancy vary in different
tumours and such difference might also be related to
different methods applied [9]. In a recent study, it is
described that alternative splicing and/or posttranslational modification of the molecules can potentially
impair the detection of those molecules by antibodies
and consequently result in high level of false negative
immunostaining [25]. Taking it in consideration, we
decided to perform IHC, as the method is easy and
constant reliable positive results are obtainable with
CD44 isoform antibodies, which are of our prime interest.
Our result has demonstrated that expressions of different isoforms of CD44 in tissues are not dependent on
the expression of the standard form, which was expressed
in all the samples. The antibody against CD44vl0
stained all the samples as well. Two of the isoforms were
differentially expressed and seemed to be related to
benign tumours. CD44v6 has been shown in some
tumours to be positively correlated to malignancy and
shorter survival [13, 16, 26, 27], however, it was also
found to correlate to a benign phenotype in other
tumours [14, 28, 29]. Expression of CD44v9 was also
associated to a benign phenotype. Our group has found
similar results in a study of lung carcinoids [30]. Expression of other isoforms did not differ between benign and
malignant tumours. CD44v6 positive patients had a
higher survival probability (Figure 2). We could not
show any statistically significant difference of survival
between CD44v6 positive and negative patients, possibly
due to the small number of patient. This should be
confirmed in a larger patient material.
In this study, we found positive immunoreaction to
almost all specimens with a new antibody against
CD44s. This might be explained by the development of
new and more specific antibodies. The serial sections of
normal human pancreas were stained with CD44s and
chromogranin-A, which showed the same pattern of
staining as reported in a previous study [18]. Pancreatic
islets were positively stained with chromogranin-A and
negative with CD44s, whereas, ductular and acinar cells
were positive with CD44s. The expression of CD44
299
References
Survival Pnobability
1 - -n
0,8"
L.
(CD44v6 +ve)
0,6 "
'
0,4 -
1
(CD44v6 -ve)
0,2 "
0 "
0
20
40
60
80 100 1 2 0 1 4 0 160 180
Time (months)
Figure 2. Kaplan-Meier curve demonstrating increased survival
in endocrine pancreatic tumours with CD44v6 immunopositivity
(P = 0.0822).
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Experimental studies have indicated that the adhesion
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might act synergistically to prevent malignant transformation.
Our results indicate a role of CD44 variant isoforms in
the malignancy and progression of EPTs. The expression
of CD44 isoforms v6 and v9, observed by immunostaining might be used as a prognostic marker in combination to the other markers in a clinical tumour biology
program.
Acknowledgements
Swedish cancer research foundation, Swedish medical
research council are acknowledged for financial support.
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Received 15 November 1999; accepted 20 January 2000.
Correspondence to.
Prof. K. Oberg
Department of Internal Medicine
University Hospital
S-751 85, Uppsala
Sweden
E-mail. Kjell.Oberg@medicin.uu.se