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Prognostic Value of CD44 Variant 6 in Laryngeal Epidermoid Carcinomas
Gülnur Güler, MD;
Sarp Saraç, MD;
Ay egül Üner, MD, PhD;
Erdem Karabulut, MD;
Aye Ayhan, MD, PhD;
Ogawa Hiroshi, MD
Arch Otolaryngol Head Neck Surg. 2002;128:393-397.
ABSTRACT
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Background CD44 variant exon 6 (v6) belongs to a family of transmembrane glycoproteins
involved in cell adhesion.
Objectives To determine the prognostic role of CD44v6 in laryngeal cancer and to
examine its relation with other clinicopathologic prognostic factors.
Design A retrospective cohort study was designed with 93 laryngeal cancer cases.
They were selected randomly from patients treated with laryngectomy between
January 1, 1983, and December 31, 1993.
Setting Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Patients The ages of the patients ranged from 31 to 73 years. Eighty-eight patients
were men and 5 were women. Three had stage I, 33 had stage II, 27 had stage
III, and 30 had stage IV disease at the time of surgery.
Intervention Histological sections of tumors and metastatic lymph nodes were reevaluated
for several histopathological factors. Sections were stained using anti-CD44v6
monoclonal antibody by immunohistochemical methods.
Results CD44v6 expression was seen only in the lower one third of the normal
squamous epithelium but in all layers of dysplasia and in situ carcinoma.
Besides a general evaluation of tumor staining, immunostaining was evaluated
separately for cell groups located in the center of neoplastic islands (nonbasal cells), at the periphery of the neoplastic islands
(basal cells), and at the infiltration zones (marginal cells). Decreased disease-free survival was noted
when there was extensive staining in the general evaluation and in cases with
extensive staining in marginal and nonbasal cells (P
= .03). Using Cox regression analysis, the greatest dimension of the largest
metastatic lymph node and extensive expression of CD44v6 in nonbasal tumor
cells were independent prognostic factors.
Conclusion Our results suggest that CD44v6 expression is an important prognostic
factor in laryngeal cancer.
INTRODUCTION
THE TNM classification system is widely used to determine the stage
and appropriate therapy in laryngeal epidermoid carcinomas.1
However, it is well known that tumors in the same stage may show different
biological behavior.2 For this reason, it is
worthwhile to investigate new prognostic factors in laryngeal cancer.
The CD44 gene, located on chromosome 11p13,
encodes a large family of transmembrane glycoproteins. It is composed of at
least 21 exons. Various isoforms of CD44 protein are produced via alternative
splicing. Ten exons (1-5 and 16-20) are expressed in all tissue types, producing
standard CD44 protein. The remaining 11 exons are added to form variant protein
isoforms.3-4 CD44 variant protein
6 (CD44v6) is produced by the insertion of exon 11 to the standard form. This
variant form was first identified in rat pancreatic carcinoma cell lines and
was found only in their metastatic clones.5
Many investigations have been performed to determine the prognostic
value of CD44v6 in various human tumors. CD44v6 overexpression correlated
with a worse prognosis in colorectal carcinoma, breast carcinoma, pancreas
carcinoma, and non-Hodgkin's lymphoma.6-7
Studies3, 8-10
that show no correlation or a positive correlation between CD44v6 overexpression
and prognosis in different types of human malignancies have also been reported.
The purpose of our study was to investigate the prognostic significance of
CD44v6 expression in laryngeal epidermoid carcinomas.
MATERIALS AND METHODS
Ninety-three case records of laryngeal epidermoid carcinoma diagnosed
between January 1, 1983, and December 31, 1993, were retrieved from the archives
of the department of pathology. Various clinical features and therapy for
these patients are summarized in Table 1.
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Table 1. Clinical Features of the 93 Patients*
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All the sections were reexamined, and histopathological factors of the
primary tumor and metastatic lymph nodes were reevaluated. Separate grades
were assigned to each tumor based on nuclear, structural, and keratinization
features. Dimensions of the tumor, tumor depth, histologic location of deepest
invasion, infiltration pattern, presence of lymphovascular and perineural
invasion, presence of dysplasia or in situ carcinoma in tumor margins, growth
pattern of tumor, ulceration, necrosis, degree of cellular reaction against
the tumor, mitotic activity in the tumor, presence of metastatic lymph nodes,
number of metastatic lymph nodes, diameter of the biggest metastatic lymph
node and metastatic focus, and presence of extracapsular invasion were considered.
A representative block was chosen for each case, and immunohistochemical
staining was performed using a streptavidin-biotin-peroxidase complex procedure.11 After deparaffinization and dehydration with xylene
and alcohol, 4-mm-thick sections were incubated with 1% hydrogen peroxide
in methanol for 15 minutes and immersed in phosphate-buffered saline for 5
minutes. For reducing unspecific background staining, blocking with normal
serum diluted 1:40 in phosphate-buffered saline at pH 7.2 containing 1% bovine
serum albumin for 20 minutes was performed. Sections were treated by microwave
3 times for 5 minutes each in citrate buffer (pH 6.0) at 620 W power. Subsequently,
they were incubated for 1 hour with a 1:200 dilution of antihuman CD44v6 monoclonal
antibody (Clone VFF-18; Bioproducts, Heidelberg, Germany). Subsequent to washing
twice with phosphate-buffered saline at pH 7.2 for 5 minutes, biotinylated
goat antimouse immunoglobulin (DAKO, Glostrup, Denmark) was applied at a dilution
of 1:100 in phosphate-buffered saline with 1% bovine serum albumin at pH 7.2
for 30 minutes, followed by peroxidase-conjugated streptavidine in a 1:100
dilution medium for 30 minutes. Finally, peroxidase activity was visualized
by diaminobenzidine, followed by counterstaining with hematoxylin. In negative
controls, the primary antibody was replaced by dilution medium. Normal squamous
epithelial staining in histologic sections was used as an internal positive
control. Two pathologists, blinded to clinical results, evaluated immunostaining
simultaneously. Staining properties of normal squamous epithelium, dysplastic
epithelium, and in situ carcinoma were noted. The entire section was evaluated
for immunohistochemical staining, which corresponds to at least 20 low-power
fields. The proportions of positive neoplastic cells were evaluated in tumors
and metastatic lymph nodes. Immunostaining was judged as negative ( 10%),
focally positive (11%-89%), or extensively positive ( 90%). Immunostaining
of neoplastic cells was appraised generally and separately as basal cells, nonbasal cells, and marginal cells (see the "CD44v6 Immunohistochemical Study" subsection
of the "Results" section).
 2 Test, Fisher exact test, and independent sample t test were used to evaluate the data obtained from this
study. The patients' disease-free survival data (DFS) were used to determine
the possible correlation between the histopathological criteria, CD44v6 expression,
and prognosis of the cases. Survival curves were constructed using the Kaplan-Meier
method. The statistical significance of these data was analyzed by the log-rank
test. Variables affecting survival were analyzed by the Cox proportional hazards
regression model.
RESULTS
CLINICAL DATA
The ages of the patients ranged from 31 to 73 years (mean, 52 years).
Eighty-eight patients (95%) were men and 5 (5%) were women. Clinical staging
according to the criteria of the American Joint Committee on Cancer12 revealed that 3 (3%) patients had stage I disease,
33 (36%) had stage II, 27 (29%) had stage III, and 30 (32%) had stage IV.
Patients had been followed up for at least 2 years, with a median follow-up
of 33.4 months.
From the evaluation of clinical data, lymph node status and stage of
the disease had a statistically significant relation with DFS (P = .01 and P<.01, respectively). In 14
of 43 patients in whom lymph nodes were enlarged, histological examination
did not prove metastasis, giving a false-positive rate of 33%. In contrast,
metastasis was seen histologically in 16 of 50 patients in whom lymph nodes
were not palpable, giving a false-negative rate of 32%. Age, sex, and clinical
T value did not have a statistically significant relation with DFS.
There was a statistically significant difference in DFS between patients
who received no additional therapy and those who received additional therapy
in the form of chemotherapy or radiotherapy (P<.01).
HISTOPATHOLOGICAL FACTORS
The tumors were divided into 2 groups based on the pattern of invasion.
The tumors with pattern 2 had infiltration as 1-layered cords or single tumor
cells. All the remaining tumors were considered under pattern 1. According
to these criteria, invasion patterns of the tumor correlated significantly
with DFS (P = .04), and the presence of perineural
invasion had a borderline significant relation with DFS (P<.06). Existence of histopathologically proven node metastasis
was highly significant (P<.01). For this reason,
other factors related to lymph node involvement were examined only in the
group of patients who had lymph node metastasis. Even after elimination of
the importance of histopathologically proven lymph node metastasis, the number
of metastatic lymph nodes (P = .02) and the greatest
dimension of the largest metastatic lymph nodes (P
= .01) had statistically significant prognostic value.
CD44v6 IMMUNOHISTOCHEMICAL STUDY
In the immunohistochemical study, normal squamous epithelium showed
membranous CD44v6 expression only in the lower one third. In contrast, in
dysplasia and in situ carcinoma, all cell layers showed positive staining
(Figure 1). In some tumors, the
cell groups in the center of the neoplastic islands (nonbasal
cells) stained less intensely compared with the basal cells that were located at the periphery of the neoplastic islands
(Figure 2). There was also more
intense staining of the neoplastic cells located at the infiltration zone
of the tumor (marginal cells) (Figure 3). In addition to a general examination, immunostaining
of so-called basal, nonbasal, and marginal cells was evaluated separately.
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Figure 1. CD44 variant exon 6 expression
in normal and neoplastic squamous epithelium (hematoxylin-eosin, original
magnification x400).
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Figure 2. A, Nonbasal cell negativity in
tumor with CD44 variant exon 6 (CD44v6, original magnification x200).
B, Extensive CD44v6 expression in nonbasal cells (CD44v6, original magnification
x100).
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Figure 3. Extensive expression of CD44 variant
exon 6 in marginal tumor cells (CD44v6, original magnification x100).
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Statistical analysis in the general examination revealed that extensive
staining with CD44v6 correlated with a significant decrease in DFS (P = .03). Furthermore, a similar decrease in DFS was noted
when the CD44v6 staining was extensive in marginal and nonbasal cells (P = .03 and P = .02, respectively)
(Figure 4).
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Figure 4. CD44 variant exon 6 (v6) positivity
in nonbasal tumor cells and disease-free survival (DFS).
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In the immunohistochemical study, the mean proportion of staining of
the neoplastic cells was 70% in primary tumors and 85% in the metastatic lymph
nodes. The proportion of lymph node metastasis staining was higher in the
primary tumor staining, but this difference was not statistically significant
(P = .10).
When the factors having a significant relation with DFS in univariate
analyses (Table 2) were evaluated
in Cox regression analysis, the greatest dimension of the largest metastatic
lymph nodes and extensive expression of CD44v6 in nonbasal tumor cells were
independent prognostic factors (Table 3). However, in the patients who received chemotherapy or radiotherapy,
the statistically significant effect of CD44v6 expression in nonbasal cells
on DFS was no longer apparent (P>.05).
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Table 2. Mean Values of Staining Proportion
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Table 3. Multivariate Analyses of Prognostic Factors for Disease-Free
Survival: Summary of Stepwise Results
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COMMENT
Overexpression of CD44 in adenomatous polyps and in the transitional
zones near the tumor reported in colon cancers supports the view that expression
of the CD44 variant forms begins early in colorectal carcinogenesis.13-14
Likewise, in our study, in normal squamous epithelium of the larynx,
positive CD44v6 immunostaining could be seen only in the lower one third of
the epithelium, with a change in the pattern in dysplasia and in situ carcinoma.
All cell layers of dysplastic epithelium and in situ carcinoma stained positively
in the cell membranes using CD44v6 antibody. Similar findings were reported
in 2 other previous studies9-10
of laryngeal cancer, suggesting its abnormal expression also begins early
in the carcinogenesis of cancer of the larynx.
In the literature, it has been reported that the overexpression of CD44v6
correlated with a worse prognosis in various cancers, such as colon, breast,
gastric cancer, and non-Hodgkin's lymphoma.3-4,6-7
In contrast, other studies3, 8-10,15-16
have shown that overexpression of CD44v6 was associated with a better prognosis
in adenocarcinomas of the lung, neuroblastomas, squamous cell carcinomas of
the head and neck, cervical cancer, and transitional carcinomas of the bladder.
CD44v6 immunostaining was investigated in laryngeal cancer in 2 previous reports.
In one of them, increased CD44v6 expression was associated with a longer survival.9 In the other one, Ostwald et al10
reported that they could not find any relation between CD44v6 expression and
metastatic behavior.
Down-regulation of CD44v6 expression during malignant transformation
of squamous tissues was reported using a different anti-v6 antibody (Var3.1).17-18 Similarly, Roye and colleagues19 suggested that overexpression of CD44v6 may be involved
in the development of esophageal dysplasia and carcinoma, but its expression
was decreased in poorly differentiated esophageal epidermoid carcinoma. van
Hal et al20 investigated head and neck epidermoid
carcinomas and cell lines using 3 different anti-v6 antibodies (U36, U39,
and VFF-18). Immunohistochemistry was performed on tumors and cell lines,
and v6-encoding splice variants were also characterized by screening a complementary
DNA library of human head and neck squamous carcinoma cell lines, using reverse
transcriptase polymerase chain reaction. In their investigation, there was
marginal or no down-regulation of CD44v6 in malignant tissue samples.
Although these discrepancies may be attributed, in part, to the differences
in immunohistochemical methods and primary antibodies used, they may also
be related to the methods of evaluation. For this reason, we tried to use
a uniform evaluation system based on location and proportion of immunopositive
cells. Furthermore, the different observations obtained in different studies
may also stem from the heterogeneity of various clinical features, such as
the location of the tumor and therapy of patients in these studies.
Disease-free survival decreased significantly when the proportion of
immunopositive nonbasal cells was 90% or greater, and this factor was an independent
prognostic factor. This finding suggests that abnormal expression of CD44v6
may also have a role in the progression of laryngeal epidermoid carcinomas.
In cases showing a staining pattern similar to that of normal squamous epithelium,
the prognosis was better than in those with extensive nonbasal staining. Although,
when all cases were included, the extensive nonbasal staining was an independent
prognostic factor, such an effect was no longer apparent in the patients who
received additional therapy. This finding suggests that patients with extensive
nonbasal CD44v6 expression may benefit from additional radiotherapy or chemotherapy.
If this series had included only the patients who did not receive additional
therapy, the significance of CD44v6 expression on DFS probably would have
been more pronounced. In the previous 2 reports that dealt with CD44v6 expression
in laryngeal cancer, different staining patterns in nonbasal and basal cells
were also noticed, but they were not evaluated separately.9-10
Lipponen et al15 used a similar method
for evaluation of immunohistochemical staining of CD44v6 in transitional cell
carcinomas of the bladder. Using a 15% cutoff value for positivity without
performing extensive staining, they documented survival in patients with CD44v6-positive
nonbasal cells.
In metastatic lymph nodes, although not statistically significant, the
percentage of tumor cells expressing CD44v6 was higher compared with that
in their primary tumors, indicating a further role in progression. Similarly,
CD44v6 was expressed in 80% of the primary tumor samples, but in 100% of the
metastatic tumors in colon cancer.21
Another finding of our study was a significantly shorter DFS that was
associated with extensive CD44v6 staining in marginal tumor cells. Although
we could not find any related finding in the literature, Sugino et al22 reported a correlation between the gradual loss of
standard CD44 protein expression and the invasive stage of the tumor in bladder
carcinoma. Expression of the structurally complex CD44
gene in cancer may be modulated by microenvironmental factors. Accordingly,
expression patterns may change as the invading neoplastic cells encounter
different mesenchymal components in the microenvironment.23
In uterine cervical cancer, loss of expression of CD44v6 was also noticed
in advanced invasive tumors.24 However, we
could not find any relation between extensive CD44v6 staining in marginal
tumor cells and the depth of invasion.
In conclusion, an abnormal expression pattern of CD44v6 seems to begin
early in the carcinogenesis of laryngeal epidermoid carcinomas. As the expression
pattern deviates from that of normal squamous epithelium, the abnormality
in expression increases, with faster progression of the tumor and a significantly
shortened DFS. In the patients who received additional therapy, the effect
of extensive CD44v6 expression in nonbasal cells was no longer obvious. This
suggests that patients with this adverse prognostic factor may benefit from
additional therapy.
AUTHOR INFORMATION
Accepted for publication September 19, 2001.
This investigation was supported by Hacettepe University research funds,
Ankara, Turkey.
Presented as a poster at the 59th Annual Meeting of the Japanese Cancer
Association, Yokohama, Japan, October 4-6, 2000.
Reprints not available from the authors.
From the Departments of Pathology, Faculty of Medicine, Hacettepe University,
Ankara, Turkey (Drs Güler, Saraç, Üner, Karabulut, and Ayhan),
and Seirei Mikatabara Hospital, Hamamatsu, Japan (Dr Hiroshi).
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