Case 9 - 838-01 51 year-old female with central back
lesion biopsied to rule out squamous cell carcinoma.
Diagnosis: Malignant melanoma, 3.21mm, level IV, with ulceration and
Case 9 - Figure A - (intermediate power micrograph): In this vertical growth phase melanoma, tumor cells extend around a blood vessel in an abluminal disposition scaffolding along the outside of the basement membrane zone of the blood vessel.
Case 9 - Figure B - Intermediate to high power micrograph of the vascular field from figure 1.
Case 10 - C02-954 61 year-old male with a lesion on the
right chest biopsied by shave technique.
Diagnosis: Malignant melanoma, 4.05mm, level IV, with ulceration, vascular and
lymphatic invasion, 34 mitoses, mm2
Case 10 - Figure A - A low power micrograph shows the base of the vertical growth phase component of this deeply invasive melanoma. There is angiolymphatic plugging by cohesive groups of neoplastic melanocytes.
Case 10 - Figure B - This blood vessel structure is distended by malignant melanocytes which are admixed with red blood cells.
Case 10 - Figure C - An intermediate power micrograph shows multiple involved blood vessels.
The propensity for melanoma to migrate along anatomical structures such as nerves (neurotropism) and
skin appendages has been recognized as a common phenomenon for many years. On the other hand this same
capacity of melanoma to migrate along the external surfaces of blood vessels and lymphatics has received
almost no attention in the literature. The origins of this potential mechanism of tumor dissemination
were perhaps first noted in the 18th century French medical literature. In his original use
of the term "metastasis" Recamier specifically referred to the spread of tumor cells along the external surfaces of vascular channels rather than within them [inadventently he was misquoted by future authors]. Subsequently in
his historic paper laying the foundations for surgical margins for melanoma in 1907 W. Sampson Handley
referred to Borst who had noted " the tendency of melanotic sarcoma to spread along the perivascular
tissues immediately outside the blood-vessels". According to Borst this attraction of melanoma cells to
blood vessels resulted from a "chemiotaxis", i.e., that blood was a necessary food for the production of
melanin. Handley explained the phenomenon as anatomical since lymphatic vessels are in close proximity
to arteries and veins, and he believed that melanoma initially disseminated by intra-lymphatic spread.
However, Handley's observations were based on the study of a single lymph node metastasis and the
regional spread of tumor from that lymph node, rather than from a primary melanma.
In recent years Lugassy and Barnhill have proposed for the first time that an important mechanism of
melanoma metastasis may be via this of migration of tumor cells along the external surfaces of vessels or
"Extravascular migratory metastasis". This hypothesized mechanism of tumor spread has been based on
ultrastructural and immunopathological studies; in the latter melanoma cells are closely apposed to the
external surfaces of the endothelial cells of blood vessels in a pericyte-like
location. Ultrastructurally the melanoma cells are linked to the endothelium by an amorphous
matrix confirmed to contain laminin (not organized in a basement membrane) by immunohistochemistry. The
latter morphological structure has been termed the "Angio-tumoral Complex". According to this proposed
mechanism, tumors cells begin the process of local spread by competing with pericytes for the
periendothelial position (of pericytes) or pericyte-like location for migration along the external
surfaces of vessels.
Using immunhistochemistry, expression of the laminin b-2 chain is observed in an angiocentric pattern
around melanoma tumor microvessels. Given the role of specific laminins in angiogenesis, protease
induction,migration, and metastasis, we have suggested that laminin or a closely related extracellular
matrical substance may play a significant role in this mechanism of tumor spread.
Lugassy and Barnhill have proposed that the histomorphological counterpart of the Angio-tumoral
Complex is angiotropic melanoma. Angiotropic melanoma has been referred to and reported anecdotally in
the literature, more as a curiosity than as an important biological phenomenon. However, the authors
have recently drawn attention to the importance of angiotropism as a biological phenomenon and prognostic
factor in localized melanoma and as the likely correlate of extravascular migratory metastasis.
In the author's experience, angiotropism is observed much more frequently than vascular invasion,
e.g., in a series of 650 consecutive invasive melanomas, the frequency of vascular invasion was
0.00014%. Vascular and/or lymphatic invasion, i.e., the presence of tumor aggregates within the latter
channels, has been thought to be a direct manifestation of metastasis in progress and accordingly a
prognostic factor. In fact, the observation of vascular/lymphatic invasion in tissue sections is
exceedingly rare as mentioned above and often an artifact which can be attributed to tortuous vascular
channels folding back into the vascular lumina and also tissue shrinkage resulting in the appearance of a
(false) vascular space. Thus at least in some instances the tumor aggregates are in fact external to the
vascular/lymphatic channel rather than being intra-luminal. As result we have questioned the practical
value of observing vascular invasion as prognostic factors in cutaneous melanoma.
We have studied the prognostic significance of angiotropism as a qualitative parameter, i.e., one that
is recorded as present or absent versus a continuous variable such as numbers of microvessels which lends
itself to quantification,in a series of patients with primary cutaneous melanoma and documented
metastasis matched with a similar group of patients with non-metastasizing primary melanomas. 80
patients with one or two representative slides and definite outcomes or follow-up were enrolled in the
study. The 80 patients comprised two groups: 1) 40 patients with melanomas metastasizing to regional
lymph nodes, visceral sites, or both and 2) 40 patients with non-metastasizing melanomas and long-term
disease-free follow-up (range 5 to 22 years, mean= 10.77). Another component of the study involved the
matching of patients from the two groups above for the following major prognostic factors: tumor
thickness in mm (usually allowing no more than a 20% difference); age in years (usually no more than a
decade difference); gender, i.e., males were matched with males and females with females; and anatomic
site matched for three major regions: head and neck, trunk, and extremities. 26 pairs of patients
resulted from this matching. In general only one or two slide(s) from each melanoma was available for
review, and this slide or slides was comprehensively assessed by a single observer (RLB) for angiotropism
and vascular/lymphatic invasion without knowledge of patient outcome. All tissue sections on each
microslide or slides were systematically studied microscopically for evidence of angiotropism (as defined
below) at the advancing front of the melanoma and up to 1 to 2 mm beyond the main tumor mass.
Angiotropism is defined as multiple melanoma cells singly disposed along or in aggregates closely
opposed to the external surfaces of (and not within) microvessels and/or lymphatics at the advancing
front and/or some distance (1 to 2 mm) away from the main part of the tumor. Angiotropism was graded as
1) absent: absence of tumor cells clearly cuffing vessels, 2) equivocal: a single focus of tumor cells
clearly cuffing a vessel, or 3) definitely present: two or more foci of tumor cells clearly cuffing a
vessel. Angiotropic melanoma cells were recognized by their unequivocal similarity to nearby melanoma
cells; if there was any question about the identity of the cells cuffing vessels the case was scored as
negative for angiotropism.
Among the 40 pairs of patients, there were no significant differences with respect to age (mean 50.7
yrs. for vs. 53.0 yrs. for patients without metastases, p=0.543, Student's two-tailed T-test); however,
patients with metastasizing melanomas had thicker melanomas versus those without metastases (2.2mm vs.
1.6mm, p=0.016). The 40 patients with metastasizing melanomas showed definite angiotropism in 16 cases
and equivocal angiotropism in 5 cases versus no definite angiotropism in any case and equivocal
angiotropism in 6 cases among the 40 melanomas without metastasis (p=0.00005, Fisher's exact test).
Melanomas with angiotropism had a mean thickness of 2.3mm and thus were significantly thicker than
melanomas without metastases (p=0.0058). We found vascular/lymphatic invasion in only a single patient
who had metastatic melanoma and also exhibited angiotropism. When the 26 matched pairs of patients were
examined, there were no differences in age and tumor thickness. However, 7 patients with metastatic
melanoma demonstrated definite and 3 equivocal angiotropism versus no definite angiotropism and only 2
melanomas with equivocal angiotropism in the group without metastases (p=0.008). Immunostaining of the
five cases with S 100 protein enhanced the recognition of angiotropic melanoma cells.
Angiotropism as defined here is more commonly observed histopathologically than vascular/lymphatic
invasion in melanoma. In addition angiotropism can be easily recognized by light microscopy in routine
tissue sections of melanoma. The use of S 100 protein may facilitate the recognition or confirmation of
angiotropism, particularly for those not experienced with this phenomenon. Our results strongly suggest
that angiotropism is an important prognostic factor correlating with metastasis.
The extravascular migratory metastasis proposed for melanoma has strong analogies with the migration
of neoplastic glial invasion of the nervous system. In order to compare our hypothesis of extravascular
migration for melanoma with the migration of glioma cells, we have undertaken experiments using the B16
murine melanoma cell line and the GL261 murine glioma cell line. These two cell lines have been used in
an in vivo murine brain tumor model. Melanoma cells were injected intracranially into mouse brains
utilizing stereotaxy on day 0. Mice were sacrificed on days 10, 15 and 20 post tumor implantation (five
mice for each time point and tumor type).
In the sections of brain tumors, both B16 and glioma cells were observed along the choroid plexus and
along the abluminal side of vessels, where they occupied a pericytic location in a pattern analogous to
the angio-tumoral complex. A clear progression along these structures was noted from the 10th to the
20th day after tumor cell injection. Similar findings were observed with both tumor cell types, but
melanoma cells were progressing more rapidely than glioma cells. There was prominent immunostaining for
laminin between tumor cells and both the choroid plexus and vascular endothelium. Laminin formed a
lattice around the vessels, with projections into the tumor mass. These data suggest the migration of
both glioma and melanoma cells along the choroid plexus and the abluminal surface of vessels.
In order to study the relationships between human melanoma cells and endothelium, we have performed
studies on the behaviour of human metastatic melanoma cells cultured in the presence of capillary-like
structures in vitro.
For studies on melanoma cell/endothelial tubule interactions, human endothelial cells (HMEC-1) were
added to Matrigel coated wells of a 96-well plate. The endothelial cells form connected tubules
representing capillary-like structures within 24 hours. Human melanoma cells were then plated in the
presence of these newly formed vascular tubules. Time-lapse video microscopy recorded the cell movements
during the next 24 hours. The same experiment repeated with melanoma cells that were preincubated with
the fluorescent vital stain confirmed the pericytic-like angiotropism of the fluorescent melanoma cells
along the tubules formed by the endothelial cells. After 6 hours, 30 to 50% of tumor cells were
elongated along the endothelial tubules. After 24 hours, all melanoma cells from the different melanoma
cell lines were localized along the external surface of the vascular tubules, occupying a pericytic-like
location in a pattern analogous to the angio-tumoral complex. These observations of pericyte-like
angiotropism of melanoma cells in vitro support the in vivo observation of melanoma cells along the
abluminal, mesenchymal surface of vessels, i.e., extravascular migratory metastasis in vivo.
In order to model in vivo invasion in its complexity, we have recently developed ex vivo and in vivo
model systems to study melanoma cell interactions with vessels. Using melanoma cell lines stably
expressing Green Fluorescent Protein (GFP), we have demonstrated the migration of fluorescent melanoma
cells along the ablumimal surface of vessels.
Ex vivo, we have developed a dynamic model of melanoma cells cocultured with vessels that have
sprouted from rat and chick aortic rings.In vivo, using the chorioallantoic membrane assay, we have
established an in vivo assay to study tumor cell /vessel interactions.
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