ARTICLE
Auteur(s) : Pascale Quatresooz, Claudine
Pierard-Franchimont, Philippe Paquet, Gérald E Pierard
Department of Dermatopathology, University Hospital
of Liège, B-4000 Liège, Belgium
accepté le 5 Janvier 2010
One current classification of skin malignant melanomas (MM)
refers to their clinical growth rates [1, 2]. Fast-growing MM are
typically characterized by a vertical growth pattern and an
estimated rate of increasing thickness reaching about
0.5 mm/month. They have a worse prognosis compared to both
slow-growing MM and growth-stunted MM, which are commonly confined
superficially in the skin [1-5]. In fact, this concept is related
to the histological distinction between the radial (superficially
spreading) growth phase and the vertical growth phase (invasion of
the dermis). Fast-growing MM usually look papular to nodular, and
they are commonly amelanotic and pinkish [5].
In many instances, bioinstrumental measurements have proven
their superiority in objectivity and accuracy over subjective
clinical gradings. The objective determination of skin colors is
conveniently performed using reflectance colorimetry in the CIELAB
color space system [6]. This type of evaluation of fast-growing MM,
to characterize their particular hue, has never been reported.
The only recognized microscopic characteristic of fast-growing
MM is the extended proportion of their growth fraction [5, 7-11].
This proliferative activity is likely related to the metastatic
risk of these neoplasms [9-11]. The concept of intravascular
dissemination of cancer cells represents a central paradigm
explaining the metastatic process [12]. Accordingly, metastatic MM
cells are thought to reach lymph nodes and other sites through the
lumen of lymphatic and blood vessels from where they possibly
extravasate. It was previously assumed that neoplastic cells in
contact with the outer portion of vessels were in the process of
intravasation or extravasation, even though there was little
morphologic and experimental evidence supporting such assumption.
Of note, the detection of MM cells in peripheral blood by reverse
transcriptase-polymerase chain reaction (rt-PCR) was not recognized
as a predictor of overt metastasis and poor clinical outcome [13].
Thus, other tissue-based mechanisms of neoplastic cell migration
likely contribute to the spread of MM cells. In particular, MM
cells have the capacity to migrate inside the peritumoral stroma of
the dermis [14]. Yet another restricted migration path resides in
the periendothelial matrix along the external portion of blood
vessels and lymphatics. This process has been documented under the
heading of angiotropism [15-21]. Such a process has been defined
histologically [19-21] by (a) unequivocal MM cells cuffing the
external surfaces of the endothelium of microvascular and/or
lymphatic channels, (b) MM cells linearly aligned or clustered in
discrete aggregates in at least 2 or more foci at the
advancing front edge of the MM or in the nearby tissue, usually
within 1 to 2 mm of the primary MM, and (c) no evidence
of intravascular or intralymphatic MM cell aggregates. Angiotropism
at the advancing front of primary MM or in nearby tissue has to be
distinguished from the entrapment of vessels by the MM.
A link between some aspects of MM biology and
neoangiogenesis/vascularity is suggested, but controversial data
have been produced [3, 22-26]. The confusion results in part from
analytical methods commonly limited to vessel section counting,
which does not represent a valid procedure [3, 22-24, 27, 28].
In this work, we explore the erythematous hue and vascularity of
fast-growing MM. The vessel size abutted to MM and the outline of
the MM front edge were assessed using morphometric analysis of
immunohistological sections combining Euclidean geometry and
fractal characterization. In addition, the MM growth fraction was
assessed. Comparisons were made between MM showing or otherwise
perivascular and interstitial MM micrometastases.
Materials and methods
Patients
A series of 32 primary MM of less than 3 months of
recognized evolution, and with a maximum thickness ranging
1.80-2.60 mm were retrieved from our files. They were selected
according to the presence (n = 16) or absence (n = 16) of MM
micrometastases in the surrounding dermis. All patients were
phototype II (n = 9) and III (n = 23), men (n = 11) and women (n =
21) aged 23-31 years and had undergone a sentinel lymph node
dissection.
High resolution colorimetry
Due to the puzzling, unusual, clinical presentation of the MM, the
colors of 17 of these neoplasms (10 with micrometastases
and 7 without metastasis) and the normal-looking skin in their
close vicinity were assessed using a Visi-Chroma VC100®
(Biophotonics, Lessines, Belgium). The explored areas were
conveniently delimited and narrowed in order to specifically focus
on the target sites [29]. Values L* and a* were measured, following
the CIELAB color space system [6]. Value L* is expressed on a scale
ranging from 0 for black to 100 for white. Chromacity a*
explored the range in the red hue on a scale extending from
100 (bright red) to 0 (white). The device provided a
spectrum of values corresponding to the digitalized segmentation of
each clinical picture. The mode of each value distribution was
recorded. Color differences (ΔL*, Δa*) were calculated between each
MM and its surrounding skin at 1-2 cm apart of the neoplasm.
Immunohistochemistry
In addition to standard microscopy, immunohistochemistry was
performed on the formalin-fixed paraffin-embedded skin excisions.
The MIB/Ki-67 immunostaining (1:100, Dako, Glostrup, Denmark)
was performed as previously described in order to reveal the MM
growth fraction represented by the ki-67 index [10, 11, 30].
MM cells were identified using antibodies directed to
S100 protein (undiluted, Dako), HMB45 (1:200, Dako) and
NKi-C3 (1:200, Dako) following previously described procedures
[10, 14, 30]. Tobetter define the outlines of the vasculature, the
endothelialstructure was highlighted using Ulex europaeus
agglutinin-1 (1:200, Dako) immunostaining as previously
described [24, 28, 31].
Angiotropism rating
Angiotropism was rated as (a) absent or equivocal (absence of MM
cells or a single spot clearly cuffing vessels), or (b) definitely
present (a few foci of MM cells clearly cuffing vessels). Other
tiny clusters of neoplastic cells disclosed at distance from the
progression edge of the primary MM and without evidence for
vascular contact were considered as interstitial micrometastases.
Those present within 2 mm from the primary neoplasm were
considered in the present study.
Morphometry and fractal analysis
Optical images were acquired using a CCD camera. The box counting
method was used as previously described [32-35] to calculate the
fractal dimension D of the deep outline of each MM. Grids of
different-size square boxes were used to cover well-contrasted
black-and-white microphotographs magnified × 660 in order to
capture details of the MM front edge. Each grid was characterized
by its box size ε. A set of 16 grids characterized by ε
increasing by 1-mm steps from 3 to 18 mm was used. These
dimensions corresponded to a range size from 4.55 to
27.27 μm on the histological slides. The entire sections were
thoroughly scanned. The number of boxes (n (ε)) necessary to cover
the given MM outlines was recorded as a function of the length of
the box edge. These data were plotted on logarithmic scales of 1/ε
against n(ε). As expected, data followed a straight line per
low-power relationship. It was defined by its negative slope S. The
fractal dimension was calculated following D = 1 - S.
The microvasculature was assessed using Euclidean geometry and
computerized image analysis (Analysis Olympus). Measurements were
performed on inverted images of tissue sections in a way that the
vessel profiles appeared as clear objects on a dark background.
Quantitative assessments of the vasculature were performed in
contiguous fields inside the 0.2 mm thick tissue zone abutted
to the deep part of the MM. Data were expressed as a relative
microvascular prolife area (RMPA, %) to the dermal area. Images
were digitized on a matrix of 512×512 pixels. After image
processing, enhancement of local discontinuities was performed
using the gradient technique [35].
Statistics
Skin color parameters and the Ki-67 indexes showed normal
distributions in the two groups of MM, exhibiting micrometastases
or not. Means and standard deviations were calculated. The unpaired
Student t-test was performed to assess statistical significances
between the two groups of MM. Calculations were made using the
package Statview 5.0 MacIntosh (Abacus Concepts Ine, Berkeley,
CA, USA).
Morphometry data did not follow Gaussian distributions. They
were expressed as medians and range. Comparisons between the two
groups of MM were made using the unpaired non-parametric
Mann-Whitney-U test. Linear regression analysis with calculation of
the coefficient r was used to compare the D values of the MM border
outlines and the corresponding MRPA. The chi-square test was
performed to compare proportions of MM showing or not lymph node
metastases. Statistical significance was reached when p was lower
than 0.05.
Results
There was no evidence for a gender and phototype influence on the
nature and colors of the examined MM.
Fast-growing MM colorimetry
Fast-growing MM globally looked pinkish to red (figure 1A, B). They showed
discrete inter-individual variations in colors. Some lesions (7/32)
were darker (lower L* value) than the peripheral skin (figure 2A), but the vast
majority (25/32) exhibited little L* difference between the
2 sites (figure
2B). The erythematous aspect (increased a* value) of the
neoplasm was discrete to moderate (figure 2C) in
12/32 MM, and more intense (figure 2D) in
20/32 MM. MM showing angiotropism were characterized by colors
(L* = 51.6 ± 11.3, ΔL* =6.3 ± 6.1, a* = 28.7 ± 6.0, Δa* = 16.6 ± 5)
undistinguishable from the MM without angiotropism (L* = 54.2 ±
9.7, ΔL* = 15.9 ± 5.3, a* = 25.4 ± 7.9, Δa* = 13.9 ± 6.4).
Angiotropism and lymph node metastases
Angiotropism was clearly identified using immunohisto-chemistry.
When present, it was associated with interstitial micrometastases
in 13/16 cases. Straightforward angiogropism (figures 3A, B) and
interstitial micrometastases were mostly found in patients with
metastasizing MM outside the skin. Indeed, 12/16 had a
positive satellite lymph node. The reverse situation was found in
cases where angiotropism and other micrometastases were equivocal
or absent. Only 1/16 exhibited a positive satellite lymph
node. The difference between the two groups was significant (p <
0.001).
MM growth fraction and angiotropism
Globally, the MIB/Ki-67 indexes were high (figure 4). They were not
statistically different in MM showing angiotropism (27.2% ± 5.0)
and MM without angiotropism (23.4% ± 6.8). Due to the low number of
MM cells involved in angiotropism and other micrometastases, it was
not possible to assess their proliferative activity with
confidence.
Vascularity beneath MM
Irregular profile densities and ramified, irregularly oriented
vessels appeared randomly distributed underneath fast-growing MM.
The vascular networks showed marked inter-individual variations in
the clustering trends of distribution. RMPA was significantly (p
< 0.05) more developed in fast-growing MM showing angiotropism
(median 9.2%, range: 3.0-23.2) than in fast-growing MM without
angiotropism (median: 6.6%, range: 2.9-9.7). No correlations (r =
.23) were found between the RMPA and the Ki-67 indexes in each
of the MM types.
Fractal dimension of the mm advancing edge
The fractal dimension D of the outline of the MM advancing edge was
significantly (p < 0.05) higher in fast-growing MM showing
angiotropism than in the neoplasm without angiotropism (figure 5). No correlations
were found between the D values and both the respective
Ki-67 indexes (r = .27) and the RMPA (r = .39).
Discussion
Chronic or intermittent sun exposure is widely accepted to have a
crucial role in the development of slow-growing MM, but does not
seem to be a key event in the pathogenesis of fast-growing MM.
Indeed, these latter MM tend to develop in individuals who
otherwise have no obvious melanoma risk factors such as a family
history of MM. These patients tend to manifest only few nevi,
freckles, and actinic keratoses [2]. Due to these conditions and
their fast growing rate, a diagnostic delay is often observed [5,
36].
Bioinstrumental assessments of skin colors using dedicated
metrological devices provide more objective, reproducibleand
quantitative information than visual scoring only [6, 29]. In our
series of fast-growing MM, value a* was unusually high, suggesting
increased vascularity. By contrast, only a modest decrease in the
L*- value indicated a restricted melanization of these neoplasms.
These findings strongly suggest that the fast-growing MM of our
series were angiogenic and exhibited discrete melanogenesis.
A series of advances in cancerology, including computed image
analysis, help in refining some prognostic factors. The
relationship between angiogenesis and MM progression remains
questionable [10, 28]. The microscopic examination of MM following
specific endothelial cell immunostaining shows variability in the
spatial distribution of the microvasculature. Indeed, both the
patterns of the tissue vascularization and the orientation and size
of the microvascular channels are variable [28]. The role of tumor
vascularity in the neoplastic progression and prognosis of
cutaneous MM is of singular importance. The understanding of the
relationship between MM cells and microvascular channels is
possibly influenced by the extent of angiogenesis, which is not by
itself a straightforward predictor for MM progression and
metastasis [22-26]. However, this concept is not firmly established
because it is difficult to translate the remarkable capacity of the
brain for identifying visual patterns into information transferable
to others. In some circumstances, the gap between the perceived
image and the transferred interpretation and knowledge may be
particularly difficult to bridge. The information about the
microscopic structure relevant for diagnosis often merely relies on
subjective pattern recognition and heuristic logic [37]. This
situation is prone to lead to an oversimplification in the
understanding of complex biological events. In the present
quantitative study, vascularity was prominent at the base of
fast-growing MM. In addition, MM angiotropism was statistically
associated with the most developed peritumoral angiogenesis. This
finding cannot distinguish among distinct pathomechanisms. It is
possible that fast-growing MM showing a peculiar propensity to
micrometastasis spreading along vessels, release angiogenic
factors. The reverse situation is also possible if the hyperplasia
of the vascular network offers a more extended periendothelial
stroma, favoring angiotropism.
The Ki-67 monoclonal antibody reveals cells engaged in the
cycle of proliferation. The procedure provides a convenient means
for evaluating the growth fraction of neoplasms. The proliferation
marker Ki-67 is a nuclear antigen expressed in all active
phases of the cell cycle of proliferation (G1,
S1, G2 and M), but absent in the resting
phase (G0). The growth fraction assessed by the
Ki-67 labeling index, is the occurrence rate of the cycling
cells, i.e. the ratio between the number of Ki-67 positive
cells to the total number of cells. There is ample evidence that
the size of the MM phase cycles and germinative pool is indicative
of the neoplastic progression [8, 10, 11, 38-40]. Indeed, previous
works indicated that MM thickness appeared correlated with the
proportion of neoplastic cells in both the S phase of proliferation
[38] or in the whole cell cycle [39, 40]. In the present study the
range of MM thickness was restricted. This situation limited the
influence of this dimensional parameter. Two main clinical
applications of proliferation markers are currently used in the
field of melanocytic neoplasms, namely the distinction between
cutaneous melanocytomas and MM, and the estimation of a clinical
prognosis in MM patients [8, 10, 11, 24, 30]. Globally, the
findings about MM growth fraction are in line with the clinical
concept distinguishing MM of high and low clinical growth rates
bearing different prognoses [1-5, 11]. In the present study, the
Ki-67 indexes of fast-growing MM were particularly high. They
had no influence on the smooth or etchy aspect of the advancing
edge of the neoplasms (D fractal dimension). Angiotropism was not
correlated with a specific range in the values of the
Ki-67 index.
The propensity for human MM to migrate along anatomic structures
such as nerves (neurotropism) and skin appendages (hair follicles
and sweat ducts) is a common phenomenon. In addition, the
extravascular migratory micrometastasis and other interstitial
micrometastases represent mechanisms by which some malignant cells
spread to nearly or more distant sites [14-21, 41]. Angiotropism
appears to be a cancer-specific marker, whereas angiogenesis and
lymphangiogenesis are not. Despite a number of studies on
microvessel density, as a correlate of angiogenesis, there are no
definitive data showing that microvessel counts represent a
prognostic factor in MM.
Interestingly, tumor cells have been reported to migrate at
rates of 0.1 to 2 μm/min, which would result in a yearly
progression of 5.2 to 105 cm. Amoeboid migration of tumor
cells could, however, achieve velocities 10 to 30 times
greater [12]. In the present study angiotropism and interstitial
micrometastases were significantly associated with the risk of
positive sentinel lymph node metastases.
In conclusion, the present study addressed for the first time in
a multi-pronged approach several clinical and microscopical
features that could participate in the micrometastatic process
linked to fast-growing MM. The most salient features are the
combination of a high Ki-67 index and increased
vascularity.
Acknowledgement
The authors appreciate the excellent secretarial assistance of Mrs.
Ida Leclercq and Marie Pugliese. This work was supported by a grant
from the “Fonds d'Investissement de la Recherche Scientifique” of
the University Hospital of Liège. Conflict of interests: none.
References
1 Lipsker D. Growth rate, early detection and prevention of
melanoma. Melanoma epidemiology revisited and future challenges.
Arch Dermatol 2006; 142: 1638-40.
2 Liu W, Dowling JP, Murray WK, et al. Rate
of growth in melanomas. Characteristics and associations of rapidly
growing melanoma. Arch Dermatol 2006; 142: 1551-8.
3 Piérard-Franchimont C, Henry F, Heymans O,
Piérard GE. Vascular retardation in dormant growth-stunted
malignant melanomas. Int J Mol Med 1999; 4: 403-6.
4 Liu W, Kelly JW, Trivett M, et al.
Distinct clinical and pathological features are associated with
BRAF mutation in primary melanoma. J Invest Dermatol 2007; 127:
900-5.
5 Bourguignon R, Giet-Lesuisse M, Arrese JE,
Piérard GE, Quatresooz P. Fast-growing melanoma. Rev Med
Liège 2009; 64: 427-9.
6 Piérard GE. EEMCO guidance for the assessment of skin
colour. J Eur Acad Dermatol Venereol 1998; 10: 1-11.
7 Boni R, Doguoglu A, Burg G, Muller B,
Dummer R. MIB-1 immunoreactivity correlates with metastatic
dissemination in primary thick cutaneous melanoma. J Am Acad
Dermatol 1996; 35: 416-8.
8 Straume O, Sviland L, Akslen LA. Loss of
nuclear p 16 protein expression correlates with increased tumor
cell proliferation (Ki-67) and poor prognosis in patients with
vertical growth phase melanoma. Clin Cancer Res 2000; 6:
1845-53.
9 Frahm SO, Schubert C, Parwaresch R,
Rudolp P. High proliferative activity may predict early
metastasis of thin melanomas. Hum Pathol 2001; 32: 1376-81.
10 Quatresooz P, Arrese JE,
Piérard-Franchimont C, Piérard GE. Immunohistochemical
aid at risk stratification of melanocytic neoplasms. Int J Oncol
2004; 24: 211-6.
11 Quatresooz P, Piérard GE,
Piérard-Franchimont C. The Mosan Study Group of Pigmented
Tumors. Molecular pathways supporting the proliferation staging of
malignant melanoma. Int J Molec Med 2009; 24: 295-301.
12 Friedl P, Wolf K. Tumour-cell invasion and
migration: diversity and escape mechanisms. Nat Rev Cancer 2003; 3:
362-74.
13 Reinhold U, Ludtke-Handjery HC, Schnautz S,
Kreysel HW, Abken H. The analysis of tyrosinase-specific
mRNA in blood samples of melanoma patients by rt-PCR is not a
useful test for metastatic tumor progression. J Invest Dermatol
1997; 108: 166-9.
14 Claessens N, Piérard GE,
Piérard-Franchimont C, Arrese JE, Quatresooz P.
Immunohistochemical detection of incipient melanoma
micrometastases. Relationship with sentinel lymph node involvement.
Melanoma Res 2005; 15: 107-10.
15 Moreno A, Espanol I, Romagosa V. Angiotropic
malignant melanoma. Report of two cases. J Cutan Pathol 1992; 19:
325-9.
16 Barnhill RL. The biology of melanoma micrometastases.
Recent Results. Cancer Res 2001; 158: 3-13.
17 Saluja A, Money N, Zivony DI, Solomon AR.
Angiotropic malignant melanoma: a rare pattern of local metastases.
J Am Acad Dermatol 2001; 44: 829-32.
18 Siegel DM, McClain SA. Angiotropic malignant
melanoma: more common than we think? J Am Acad Dermatol 2001; 44:
870-1.
19 Barnhill RL, Dy K, Lugassy C. Angiotropism in
cutaneous melanoma: a prognostic factor strongly predicting risk
for metastasis. J Invest Dermatol 2002; 119: 705-6.
20 Lugassy C, Barnhill RL. Angiotropic malignant
melanoma and extravascular migratory metastasis: description of 26
cases with emphasis on a new mechanism of tumor spread. Pathology
2004; 36: 485-90.
21 Lugassy C, Barnhill RL. Angiotropic melanoma and
extravascular migratory metastasis. A review. Adv Anat Pathol
2007; 14: 195-201.
22 Busam KJ, Berwick M, Blessing K, et al.
Tumor vascularity is not a prognostic factor for skin melanoma. Am
J Pathol 1995; 147: 1049-56.
23 Marcoval J, Moreno A, Graells J, et al.
Angiogenesis and malignant melanoma. Angiogenesis is related to the
development of vertical (tumorigenic) growth phase. J Cutan Pathol
1997; 24: 212-8.
24 Piérard GE, Piérard-Franchimont C. Stochastic
relationship between the growth fraction and vascularity of thin
malignant melanomas. Eur J Cancer 1997; 33: 1888-92.
25 Sivridis E, Giatromanolaki A, Koukourakis MI.
The vascular network of tumours, what is it not for ? J Pathol
2003; 201: 173-80.
26 Verheul HM, Voest EE, Schlingemann RO. Are
tumours angiogenesis-dependent? J Pathol 2004; 202: 5-13.
27 Zielinski KW. Practical problems in quantification of
tissue vascularisation. Acta Stereol 1998; 17: 59-72.
28 Heymans O, Blacher S, Brouers F,
Piérard GE. Fractal quantification of the microvasculature
heterogeneity in cutaneous melanoma. Dermatology 1999; 198:
212-7.
29 Hermanns JF, Piérard GE. High resolution
epiluminescence colorimetry of striae distensae. J Eur Acad
Dermatol Venereol 2006; 20: 282-7.
30 Quatresooz P, Piérard-Franchimont C,
Piérard GE. Highlighting the immunohistochemical profile of
melanocytomas. Oncol Reports 2008; 19: 1367-72.
31 Quatresooz P, Piérard GE. Immunohistochemical clues
at aging of the skin microvascular unit. J Cutan Pathol 2009; 36:
39-43.
32 Cross SS, Cotton DWK. The fractal dimension may be
a useful morphometric discriminant in histopathology. J Pathol
1992; 166: 409-11.
33 Cross SS. The application of fractal geometric analysis
to microscopic images. Micron 1994; 25: 101-13.
34 Buczkowski S, Kyriacos S, Nekka F,
Cartilier LH. Some guidelines for estimating fractal dimension
with a box-counting method in image analysis. Patt Recog 1998; 31:
411-8.
35 Uhoda I, Piérard GE, Piérard-Franchimont C,
et al. Vascularity and fractal dimensions of the
dermo-epidermal interface in gutate and plaque type psoriasis.
Dermatology 2005; 210: 189-93.
36 Betti R, Martino P, Vergani R,
Gualandri L, Crosti C. Nodular melanomas: analysis of the
casuistic and relationship with thick melanomas and diagnostic
delay. J Dermatol 2008; 35: 643-50.
37 Cross SS, Cotton DWK, Underwood JCE. Measuring
fractal dimensions. Sensitivity to edge-processing functions. Anal
Quant Cytol Histol 1994; 16: 375-9.
38 Piérard GE, Piérard-Franchimont C, Henry C,
Lapière M. The proliferative activity of cells of malignant
melanomas. Am J Dermatopathol 1984; 6: S317-S324.
39 Sparrow LE, English DR, Taran JM,
Heenan PJ. Prognostic significance of MIB-1 proliferative
activity in thin melanomas and immunohistochemical analysis of
MIB-1 proliferative activity in melanocytic tumors. Am J
Dermatopathol 1998; 20: 12-6.
40 Quatresooz P, Piérard-Franchimont C,
Piérard GE. The Mosan Study Group of Pigmented Tumours.
Molecular histology on the diagnostic cutting edge between
malignant melanomas and cutaneous melanocytomas. Oncol Reports
2009; 22: 1263-7.
41 Shaikh L, Sagebiel RW, Ferreira CMM,
Nosrati M, Miller 3rd JR,
Kashani-Sabet M. The role of microsatellites as a prognostic
factor in primary malignant melanoma. Arch Dermatol 2005; 141:
739-42.
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