ARTICLE
Auteur(s) : Levent Yildiz, Mehmet Kefeli, Oguz Aydin,
Bedri Kandemir
Ondokuz Mayis University, Medical School, Department
of Pathology
Kurupelit 55139 Samsun-Turkey
accepté le 28 Mars 2009
Malignant melanoma is relatively common and one of the most
important malignant tumors because of its very high mortality rate
[1, 2]. Familial and environmental factors play a role in the
etiology of the malignant melanoma. It may occur de nova or arise
within pre-existing melanocytic nevi [3]. Therefore benign
melanocytic lesions may be precursors for the development of
malignant melanoma and could exist together in approximately half
of cases [4]. Histological diagnosis of malignant melanoma is based
on cellular features and architectural criteria. Because some
melanocytic nevi and malignant melanomas have very similar
architectural and cytological features, differential diagnosis of
malignant melanoma from benign melanocytic lesion can be
histopathologically difficult in problematic cases. Misdiagnosis of
malignant melanoma is the second most frequent cause of malpractice
claims in the USA [5, 6]. Additional diagnostic markers may
therefore be helpful in the assessment of melanocytic lesions.
Fascin is a -55 kDa-actin binding protein. It is a
well-conserved actin regulatory protein. Actin bundles rearranged
by fascin proteins are concentrated in cell membrane protrusions,
and these protrusions provide motility of the cell [7, 8]. There
are 3 related fascin proteins in human cells. Fascin-1 is the most
common type and is found in most human tissues. Fascin-2 and
fascin-3 have limited distribution and are exclusive to the retina
and the testis [9, 10]. Fascin has been labeled an important
protein in carcinogenesis by playing an important role in cellular
motility and migratory changes in carcinogenesis by forming actin
based structures [11]. Multiple neoplasms such as skin tumors,
glial tumors and carcinomas of the pancreas, lung, breast, stomach,
esophagus and bladder express fascin [12-21].
The aim of the present study was to: (a) investigate the
expression of fascin in a group of melanocytic lesions which
included benign nevi (BN), dyplastic nevi (DN), lentigo malignas
(LM) and malignant melanomas (MM), (b) determine whether fascin
expression could aid differential diagnosis in these lesions, and
(c) investigate fascin expression in MM and metastatic malignant
melanomas (MMM).
Materials and methods
This retrospective study involved 73 cases of melanocytic lesions
of the skin, including 20 BN (4 junctional, 8 compound, 8
intradermal), 12 DN, 4 LM, 25 MM (13 of superficial spreading
melanoma, 8 of nodular melanoma, 2 of lentigo malignant melanoma
and 2 acral lentiginous melanoma), and 12 MMM (9 lymph node, 3
visceral). Primary melanomas were classified according to Clark’s
level (I, n = 0; II, n = 5; III, n = 10; IV, n = 7; V, n = 3) and
to Breslow thickness (< 1 mm, n = 5; 1-2 mm, n =
6; 2-3 mm, n = 5; 3-4 mm, n = 4; > 4 mm, n = 5).
All specimens had been routinely fixed in formalin and processed in
paraffin wax. All routine hematoxylin and eosin (H&E) stained
slides were reviewed and the histological diagnoses reconfirmed by
two pathologists. The best samples of sections stained with H&E
were selected for immunohistochemistry.
Immunohistochemical staining
Four micrometer sections were prepared from routinely processed
paraffin blocks. Slides were deparaffinized and rehydrated.
Endogenous peroxidase activity was blocked with 3% hydrogen
peroxide in distilled water for 10 min. For antigen retrieval,
the slides were heated in a microwave for 10 min in
0.01 mol/L citrate buffer solution (pH 6.0) and treated with
protein block, serum-free (Dako, Carpinteria, CA, USA) for
5 min at room temperature. Slides were then incubated with
primary antibody fascin (Fascin Ab-1, Clone 55K-2, Thermo
Scientific, USA) at room temperature for 30 min, and then with
secondary antibody for 30 min. Finally the sections were then
reacted in 3-amino-9-ethyl carbazole (AEC), counterstained with
Mayer’s hematoxylin, and mounted. Endothelial cells were used for
internal positive control.
Assessment of immunohistochemical staining
The stained slides were assessed by two pathologists without
knowledge of the pathological diagnosis and the cytoplasmic
staining was accepted as positive. Each slide was evaluated
according to staining extent and intensity. Staining extension was
assessed as the percentage of stained cells and scored
semiquantitatively, using a 0 to 4 scale for expression: 0 = no
expression; 1+ = 1-25%, 2+ = 26-50%, 3+ = 51-75%, 4+ = 76-100.
Staining intensity was categorized into three groups by comparing
the staining intensity of tumor cells with vascular endothelial
cells: 1+ = weaker than endothelial cells; 2+ = same as endothelial
cells; and 3+ = stronger than endothelial cells [15]. By adding the
staining extension and intensity scores, the combined scores were
calculated. The combined scores were then divided into 4 groups:
Negative = combined score 0; weak staining = combined score 2;
moderate staining= combined scores 3-4; strong staining= combined
scores 5-7.
Statistical analysis
The Chi-Square test and Fisher’s exact test were used to compare
fascin expression among the groups (benign nevi, dysplastic nevi,
lentigo maligna, malignant melanoma and metastatic malignant
melanoma). The Mann Whitney U test was employed to compare the
extension and intensity of fascin expression in the malignant
melanoma group, according to Clark’s level and Breslow thickness.
The SPSS system (SPSS Inc., Chicago, IL, USA) was used for
calculation and p values of less than 0.05 were considered
statistically significant.
Results
Fascin expression was seen in 19/20 (95%) BN, in 8/12 (67%) DN, in
1/4 (25%) LM, in 7/25 (28%) MM and in 3/12 (25%) MMM cases. The
frequency and combined scores of fascin expression of groups are
shown in table 1. Fascin expression was
moderate in 2/20 (10%) cases and strong in 17/20 (85%) cases in the
BN group (figures 1A,
B). Average combined score was 5.5. There was one negative
case of compound nevus. There was no difference for fascin
expression among compound, junctional and intradermal nevi. Fascin
expression was weak in 1/12 (8%), moderate in 5/12 (42%) and strong
in 2/12 (17%) cases in the DN group. Average combined score was
2.8. There was no significant difference for fascin expression
between the BN and the DN groups (p = 0.053).
In the LM group, fascin expression was seen in only one of 4
cases (25%). Fascin expression was moderate in this case. Average
combined score was 1; it was significantly lower than for benign
groups (p < 0.001), but was not statistically different from the
DN group (p > 0.05). There were very few cases in the LM group,
so any diagnostic implications are unclear.
For the MM group, fascin expression and clinicopathological
information are summarized in table 2.
Fascin expression was weak in 3/25 (12%) cases, moderate in 2/25
(8%) cases and strong in 2/25 (8%) cases in this group (figure 2). There was no
significant difference between positive and negative cases
according to Clark’s level and Breslow thickness. And also there
was no significant difference for fascin expression according to
Clark’s level and Breslow thickness in positive cases. There was a
significant difference for fascin expression between MM and BN (p
< 0.001) and MM and DN (p = 0.036). In the MMM group, fascin
expression was moderate in 2 (16%) cases and strong in 1 (8%) case
(figure 3).
Average combined score was 1.04 for primary MM and 1 for MMM (p
> 0.05). Comparisons of groups are summarized in table 3.
Table 1 Frequency and combined scores of fascin
expression in melanocytic lesions
|
BN (n = 20)
|
DN (n = 12)
|
LM (n = 4)
|
MM (n = 25)
|
MMM (n = 12)
|
|
Fascin expression
|
|
|
|
|
|
|
Positive
|
19 (95%)
|
8 (67%)
|
1 (25%)
|
7 (28%)
|
3 (25%)
|
|
Negative
|
1 (5%)
|
4 (33%)
|
3 (75%)
|
18 (72%)
|
9 (75%)
|
|
Extension scores
|
|
|
|
|
|
|
0
|
1 (5%)
|
4 (33%)
|
3 (75%)
|
18 (72%)
|
9 (75%)
|
|
1+
|
0 -
|
2 (17%)
|
0 -
|
3 (12%)
|
1 (8%)
|
|
2+
|
1 (5%)
|
4 (33%)
|
1 (25%)
|
2 (8%)
|
1 (8%)
|
|
3+
|
13 (65%)
|
1 (8%)
|
0 -
|
1 (4%)
|
1 (8%)
|
|
4+
|
5 (25%)
|
1 (8%)
|
0 -
|
1 (4%)
|
0 -
|
|
Intensity scores
|
|
|
|
|
|
|
1+
|
1 (5%)
|
4 (50%)
|
0 -
|
4 (57%)
|
1 (33%)
|
|
2+
|
6 (32%)
|
2 (25%)
|
1 (100%)
|
1 (14%)
|
1 (33%)
|
|
3+
|
12(63%)
|
2 (25%)
|
0 -
|
2 (29%)
|
1 (33%)
|
|
Combined scores
|
|
|
|
|
|
|
0 (Negative)
|
1 (5%)
|
4 (33%)
|
3 (75%)
|
18 (72%)
|
9 (75%)
|
|
2 (Weak)
|
0 -
|
1 (8%)
|
0 -
|
3 (12%)
|
2 (17%)
|
|
3-4 (Moderate)
|
2 (10%)
|
5 (42%)
|
1 (25%)
|
2 (8%)
|
1 (8%)
|
|
5-7 (Strong)
|
17 (85%)
|
2 (17%)
|
0 -
|
2 (8%)
|
0 -
|
- Average scores (0-7)
- (Mean and SD)
|
5.5 (±1.6)
|
2.58 (±2.3)
|
1 (±2)
|
1.04 (±1.9)
|
1 (±1.9)
|
Table 2 Fascin expression and clinicopathologic
information for malignant melanoma cases
|
No
|
Age/Sex
|
Melanoma type
|
Clark
|
Breslow (mm)
|
Fascin expression
|
|
1
|
67/M
|
LMM
|
IV
|
2.7
|
-
|
|
2
|
65/M
|
LMM
|
III
|
2.6
|
-
|
|
3
|
63/F
|
ALM
|
V
|
6.6
|
-
|
|
4
|
57/M
|
ALM
|
III
|
0.6
|
+
|
|
5
|
59/F
|
SSM
|
III
|
0.5
|
+
|
|
6
|
48/M
|
SSM
|
III
|
1.5
|
-
|
|
7
|
61/M
|
SSM
|
IV
|
2.5
|
-
|
|
8
|
52/M
|
SSM
|
II
|
0.7
|
-
|
|
9
|
62/F
|
SSM
|
IV
|
2.3
|
-
|
|
10
|
34/M
|
SSM
|
III
|
1.3
|
-
|
|
11
|
38/M
|
SSM
|
II
|
0.7
|
++
|
|
12
|
56/M
|
SSM
|
III
|
0.8
|
++
|
|
13
|
63/M
|
SSM
|
IV
|
1.5
|
-
|
|
14
|
74/F
|
SSM
|
II
|
1.2
|
-
|
|
15
|
47/F
|
SSM
|
II
|
1.0
|
-
|
|
16
|
30/M
|
SSM
|
II
|
1.8
|
-
|
|
17
|
50/F
|
SSM
|
III
|
2.0
|
+++
|
|
18
|
57/M
|
NM
|
IV
|
3.5
|
+++
|
|
19
|
73/F
|
NM
|
IV
|
4.7
|
-
|
|
20
|
59/M
|
NM
|
III
|
3.7
|
+
|
|
21
|
74/M
|
NM
|
IV
|
5.4
|
-
|
|
22
|
54/M
|
NM
|
V
|
5.6
|
-
|
|
23
|
53/F
|
NM
|
III
|
3.8
|
-
|
|
24
|
53/M
|
NM
|
V
|
6.5
|
-
|
|
25
|
62/M
|
NM
|
III
|
3.0
|
-
|
|
26
|
81/M
|
MMM(LN)
|
|
|
++
|
|
27
|
36/M
|
MMM(LN)
|
|
|
-
|
|
28
|
68/F
|
MMM(LN)
|
|
|
+++
|
|
29
|
63/M
|
MMM(LN)
|
|
|
++
|
|
30
|
79/M
|
MMM(LN)
|
|
|
-
|
|
31
|
39/F
|
MMM(Lung)
|
|
|
-
|
|
32
|
59/F
|
MMM(LN)
|
|
|
-
|
|
33
|
56/M
|
MMM(LN)
|
|
|
-
|
|
34
|
67/M
|
MMM(Liver)
|
|
|
-
|
|
35
|
63/M
|
MMM(LN)
|
|
|
-
|
|
36
|
76/F
|
MMM(LN)
|
|
|
-
|
|
37
|
62/M
|
MMM(Liver)
|
|
|
-
|
Table 3 P values of comparison of groups for fascin
staining
|
Groups
|
p
|
|
Benign Nevi - Lentigo Maligna
|
0.001a
|
|
Benign Nevi - Dysplastic Nevi
|
0.053
|
|
Benign Nevi - Malignant Melanoma
|
< 0.001a
|
|
Dysplastic Nevi - Lentigo Maligna
|
> 0.05
|
|
Dysplastic Nevi - Malignant Melanoma
|
0.036a
|
|
Malignant Melanoma - Lentigo Maligna
|
> 0.05
|
|
Malignant Melanoma - Metatastatic Melanoma
|
> 0.05
|
aStatistically significant.
Discussion
The diagnosis of benign, dysplastic and malignant melanocytic
lesions depends on architectural and cytological features [3].
However, distinguishing among them can sometimes be problematic
because of the difficulty of discerning the cellular and
architectural features critical for diagnosis. It is also the case
that some histological features of malignancy such as cellular
atypia, loss of maturation and mitotic activity may be seen in
benign and dysplastic melanocytic lesions [3]. Because malignant
melanoma is a highly malignant tumor and has the highest mortality
rate, its discrimination from benign lesions is essential for
patient management and survival. Therefore, it would be useful to
have additional markers to distinguish among these lesions. This
study indicated that fascin is more frequently and extensively
expressed in BN and DN than in MM.
Fascin is essential for cell-to-cell interactions, cellular
migration and cell-matrix adhesion. Fascin expression is highly
specific to cell and tissue types. Actively migrating cells express
high levels of fascin but this protein is undetectable in most
normal epithelial cells. It plays an important role in cellular
motility and migratory changes in carcinogenesis by forming actin
based structures, and it is expressed in multiple neoplasms.
Because cell motility is an important factor in the invasion and
metastasis of cancers, fascin expression usually correlates with
high grade, extensive invasion or metastasis in these tumors
[13-21]. Hashimoto et al. researched the prognostic significance of
fascin expression in gastric carcinomas and found high fascin
expression associated with poor prognosis [14]. Pelosi et al.
investigated fascin expression in 220 patients with stage I non
small cell lung carcinomas. They found high fascin expression
associated with high grade tumor and short survival [18]. Peraud et
al. investigated fascin expression in astrocytic neoplasms and they
found high fascin expression in the high grade astrocytomas (grades
III and IV) [19]. Helige et al. investigated motility, cytoskletal
actin and gap junctional communication in benign and malignant
melanoma cells in vitro [22]. They found normal melanocytes had
less motility, more highly organized cytoskeletal actin and more
vinculin plaque than malignant melanoma cells, but there was no
difference for gap junctional communication. The current study
found less fascin expression in the MM group than the BN and DN
groups. Disorganisation of the actin cytoskletons of melanoma cells
may be the reason for less fascin expression by these cells.
To our knowledge, there is only one study of fascin expression
in melanocytic tumors in the literature. Goncharuk et al.
investigated fascin expression in various skin neoplasia [12]. In
their study, 17 of 19 (89%) melanocytic nevi and 3 of 16 (19%)
malignant melanoma cases were positive for fascin. They found
heterogeneous, regional to focal fascin staining in MM cases.
Staining intensity was weak to intense in this group. All benign
nevi showed moderate to intense, diffuse fascin staining. However 2
of 4 dysplastic nevi were only focally stained. Our study results
were similar to Goncharuk’s study. We found fascin expression in 19
of 20 benign nevi. Fascin staining was moderate to strong in all
positive benign nevi cases. We also found fascin expression in 7 of
25 MM cases. Fascin staining was heteregenous and intensity ranged
from mild to strong. Our study also investigated the relationship
of fascin expression with the Clark’s level and Breslow thickness
but there were no significant differences. Goncharuk et al. stated
that indolent skin cancers have higher fascin expression than
aggressive and metastatic tumors [12]. They suggested that high
level fascin expression can be more linked to local invasiveness
than metastatic potential. We compared fascin expression between
primary and metastatic MM cases but did not find a significant
difference. There were very few cases in the MMM group in our
study. Therefore further study with larger case numbers is needed
to support their suggestion.
There are a few markers which could be used for differential
diagnosis of melanocytic lesions such as HMB-45, S-100, MART1,
Ki-67, etc. The sensitivity and specificity of these markers are
different for some melanocytic lesions [23]. Therefore, generally,
these are used with each other for melanocytic lesions. We found
statistically significant differential fascin expression exists
between benign nevi and malignant melanomas in our study. But 7 of
25 (28%) malignant melanoma cases were positive for fascin like
benign nevi. Nevertheless, we think that fascin could be used with
other melanocytic markers for differential diagnosis of melanocytic
lesions.
While normal growing tissues have no fascin expression, their
neoplastic counterparts have high fascin expression which is
associated with metastasis and poor prognosis. This situation is
contrary to that for melanocytic tumors [8, 12]. In the neoplastic
transformation of melanocytes and in metastatic processes, fascin
probably has a different role from that defined in other tumors in
the literature [11].
In conclusion, we found significantly higher levels of fascin
expression in BN and DN than in MM. Fascin may therefore be a
useful immunohistochemical marker to distinguish between these
lesions. Its diagnostic value should be further investigated in a
study with a larger number of cases.
Acknowledgements
The authors thank Gregory T. Sullivan of OYDEM, Ondokuz Mayis
University in Samsun, Turkey for proofreading an earlier version of
this manuscript. Financial support: none. Conflict of interest:
none.
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