—  SPECIALTY CONFERENCE  —

Dermatopathology

Cases 9&10 - Vascular Invasion in Primary Melanoma vs Extravascular Migratory Metastasis: Mechanisms and Morphology

Raymond Barnhill
George Washington University Medical School
Washington, DC


Click on each slide thumbnail image for an enlarged view
Clinical Histories:
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 angiotropism


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.

References

  1. Barnhill RL, Fine J, Roush GC, Berwick M. Predicting five-year outcome from cutaneous melanoma in a population-based study. Cancer 78:427-432,1996.
  2. Busam KJ, Berwick M, Blessing K, Fandrey K, Karaoli T, Fine J, Cochran
  3. AJ,WhiteWL, Rivers J, Elder DE, Wen D-RP, Heyman BH, Barnhill RL. Tumor vascularity is not a prognostic factor for skin melanoma. Am J Pathol 147:1049-1056, 1996.
  4. Recamier JCA In : Recherches sur le traitement du cancer. 1829, Gabon, Paris.
  5. Lugassy C, Escande JP. The hematogenous theory of metastasis: Recamier did not propose it. Virchows Arch. 1997,431: 371
  6. Handley WS. The pathology of melanotic growths in relation to their operative treatment. Lancet 11907;927: 996-998.
  7. Lugassy C, BP Eyden, L Christensen and JP Escande. Angio-tumoral complex in human malignant melanoma characterised by free laminin: ultrastructural and immuno-histochemical observations. J Submicrosc Cytol Pathol 1997;29: 19-28
  8. Lugassy C, L Christensen, M Le Charpentier, E Faure and JP Escande, 1998. Ultrastructural and immunohistological observations concerning laminin in B16 melanoma: is an amorphous form of laminin promoting a non hematogenous migraton of tumor cells? J Submicrosc Cytol Pathol, 30:137-44
  9. Lugassy C, L Christensen, M Le Charpentier, E Faure and JP Escande, 1998. Angio-tumoral laminin in murine tumors derived from human melanoma cell lines. Immunohistochemical and ultrastructural observations. J Submicrosc Cytol Pathol. 30(2):231-237.
  10. Lugassy C, GR Dickersin, L Christensen, T Karaoli, M Le Charpentier, JP Escande, RL Barnhill. Ultrastructural and immunohistochemical studies of the periendothelial matrix in malignant melanoma: evidence for an amorphous matrix containing laminin. J Cutan Pathol 1999;26:78-83
  11. Lugassy C, Shahsafaei A, Bonitz P, Busam KJ, Barnhill RL. Tumor microvessels in melanoma express the beta-2 chain of laminin. Implications for melanoma metastasis. J Cutan Pathol. 1999 ;26:222-226
  12. Moreno A, Espanol I, Romagosa V. Angiotropic malignant melanoma. Report of two cases. J Cutan Pathol. 1992;19:325-9.
  13. Shea CR, Kline MA, Lugo J, McNutt NS. Angiotropic metastatic malignant melanoma.Am J Dermatopathol 1995 ;17 :58-62.
  14. Saluja A. Moncy N, Zivony DI, Solomon AR. Angiotropic malignant melanoma: a rare pattern of local metastases. J Am Acad Dermatol 2001;44:829-832.
  15. Lugassy C, Kleinman HK, Fernandez PM, Patierno SR, Webber MM, Ghanem G, Spatz A, Barnhill RL. Human melanoma cell migration along capillary-like structures in vitro: a new dynamic model for studying extravascular migratory metastasis. J Invest Dermatol. 2002;119:703-704.
  16. Lugassy C, Haroun RI, Brem H, Tyler BM, Jones RV, Fernandez PM, Patierno SR, Kleinman HK, Barnhill RL. Pericytic-like angiotropism of glioma and melanoma cells. Am J Dermatopathol. 2002;24:473-478.