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Microvascular loops and networks in uveal melanoma
Microvascular loops and networks in uveal melanoma Tero Kivela, MD; Teemu Makitie, MD; Rana'a T. Al-Jamal, MD; Paivi Toivonen, MD ABSTRACT Microvascular patterns- three-dimensional architectural arrangements of microvessels and extravascular matrix in uveal melanoma - were discovered when investigators were looking for histopathological features of sufficient size to be imaged clinically. Evidence that these patterns may be formed by tumour cells and that they may be able to conduct plasma and blood as well as discovery of similar elements in other cancers make them of general importance. Of nine different patterns described, closed microvascular loops and networks have been studied most extensively. When cell type, microvascular density and nucleolar size are controlled for, these two patterns independently predict time to metastasis. In addition to visualization in tumour specimens stained with periodic acid-Schiff reagent, they can often be visualized clinically on confocal indocyanine green angiography. The presence of networks is clinically associated with probability of growth of small uveal melanocytic tumours and with the rate of regression of uveal melanoma after brachytherapy. Networks are also associated with development of exudative retinal detachment from uveal melanoma. Histopathological studies show that loops and networks are less common in tumours enucleated after irradiation and that they are frequently repeated in metastases of uveal melanoma. Avenues for immediate future research include detailed elucidation of the histogenesis of microvascular patterns and determination of these patterns in metastatic melanoma to identify new histopathological characteristics for prognostication when clinical metastases have developed.
n his 1882 monograph Das Sarcom des Uvealtractus, Fuchs 1 drew attention to a subgroup of uveal melanomas that his contemporaries called "alveolar sarcoma" and "combined sarcoma and carcinoma." He identified 14 tumours of this type among 258 uveal melanomas known to him and noted that they were particularly rapidly growing, so that enucleation became necessary an average of 10 months after the
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From the Ocular Oncology Service and Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland Correspondence to: Dr. Tero Kivela, Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, Haartrnaninkatu 4 C, PL 220, FIN-00029 HUS, Helsinki, Finland; tero.k:[email protected] Can J Ophthalmol 2004;39:409-21
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onset of symptoms, 21 months earlier than usual. Fuchs thought that these were the most malignant uveal melanomas, because 8 of the 14 patients had died from metastasis. 1 He found that histologically the metastases repeated the alveolar structure of the primary uveal tumour. In retrospect, some of the tumours that Fuchs observed may have appeared alveolar because of microvascular networks, 2-6 a matrix pattern found in uveal melanomas that occasionally is very conspicuous even on routine light microscopy (Fig. 1, A). Fuchs did not specifically link the pattern with tumour vascularization, far less discover that networks are but a special case of a novel type of tumour microcirculation that is now known as vasculogenic mimicry. 5•6 This discovery was left for other investigators to make. In 1992 Folberg and colleagues4 searched for light microscopic features of uveal melanoma that would
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Fig. !-Spectrum of microvascular networks in primary ciliochoroidal melanoma (A, D, E, F, H) and its metastases (B, C). A: Microvascular networks in amelanotic choroidal melanoma on routine hematoxylin-eosin staining. Note erythrocytes between matrix layers (arrowheads). B: Networks are better visualized after "gold standard" melanin bleaching and periodic acid-Schiff reagent (PAS) staining without counterstain. C: Identification is further aided by using dark green filter. 0: Pencil-thin networks and associated PAS-positive macrophages (arrowheads) in spindle cell melanoma. Reduplicated matrix forms networks of miniature loops (E) that enclose single epithelioid cells (arrowheads) (F). G: Thicker networks with macrophages in liver metastasis. H: Ciliary muscle bundles mimic microvascular networks. (A, F: hematoxylin-eosin; B, C, D, E, G, H: PAS without counterstain. Magnification: B, C, G: x75; A, D: xl45; E, F, H: x285.)
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Microvascular loops and networks-KiveHi et a1 be associated with survival and large enough to be visualized noninvasively. They noted that the arrangement of microvessels varied from tumour to tumour and that these patterns fell into nine categories, one of which was microvascular networks. Certain patterns were typical of nevi and low-grade uveal melanomas, 7 whereas· others predicted a high risk of metastasis after enucleation. 3.4.s-10 These investigators were the first to understand the potential implications of this observation, and so commenced the era of microcirculation in the study of uveal melanoma. A related line of research concentrated on microvascular density, which in 1991 had been linked with the risk of metastasis in breast cancer. 11 In 1996 Foss and associates 12 confirmed that high numbers of immunohistochemically labelled microvessels in areas of densest vascularization, or "hot spots," were associated with death after enucleation of uveal melanoma. Although initially challenged, the observations of both groups were later confirmed beyond reasonable doubt.l 3- 17 Although the topic of our review is microvascular loops and networks, we find it important to emphasize that we concur with Folberg and colleagues, 10 who stressed that other matrix patterns, in particular parallel with cross-linking, arcs and arcs with branching, are also associated with risk of metastasis. We focus on loops and networks because they have been most strongly and consistently associated with risk of death from metastasis of uveal melanoma and because these two are by far the most extensively studied of the nine microvascular patterns. 2-4,6-10,I5-29
These networks may provide a novel form of circulation to malignant tumours in addition to classic angiogenesis and vascular cooption. 5 Endothelial-cell-lined blood vessels can be embedded between the matrix sheets. 31 •36 Vasculogenic mimicry has subsequently been described in other cancers, such as cutaneous melanoma6•37- 39 and breast, 40 ovarian41 and prostate42 carcinoma. It has been difficult to prove conclusively the presence in human uveal melanoma of microvascular channels, which are not lined by endothelial cells, 35 although electron microscopic images have suggested that they exist. 5•6•43 In three-dimensional cultures, aggressive uveal melanoma cells capable of metastasis form PAS-positive looping and networking channels, which consist of laminin and other basement membrane components. Tracer substance can be injected through the channels.6.43 Likewise, in xenografts of cutaneous melanoma, sheets of extracellular matrix rich in laminin and collagen are deposited around clusters of tumour cells. 31 Slits between the sheets seem to conduct fluid, and they may functionally mimic lymphatic vessels. These spaces also contain macrophages. 31 These are interesting observations, because the presence of loops and networks in uveal melanoma increases the likelihood of exudative retinal detachment. 44 A melanoma that has networks is four times as likely to cause retinal detachment as a melanoma of the same size and location but without loops and networks. 44 Interstitial fluid conducted through extravascular networks may explain this association. HIERARCHY OF MICROVASCULAR PATTERNS
fORMATION OF MICROVASCULAR PATTERNS
Different opinions about the histogenesis, nature and function of the potentially perfused connective tissue patterns, which can be identified with periodic acid-Schiff stain (PAS), have led to variable terminology over time: they have been called vascular, 4 fibrovascular,l5 PAS, 20 microcirculatory30 and, most recently, extravascular matrix 31 •32 patterns. A key hypothesis, which has aroused great interest and debate, 33-35 maintains that uveal melanomas can acquire part of their circulation by generating patterned extravascular matrix channels that conduct plasma and, sometimes, blood. 5•6 The formation of these threedimensional, matrix-rich, vascular-like networks together with deregulated expression of genes typical of vascular cells is designated vasculogenic mimicry. 5•6
According to Folberg and colleagues, 10 the nine microvascular patterns form two groups: those of linear patterns ("straight," "parallel" and "parallel with cross-linking") and those of curved patterns ("arcs," "arcs with branching," "loops" and "networks"). The patterns of "silence" and "normal vessels" appear to belong to a third group that is characteristic of uveal nevi. 7 These three groups, which are interrelated, 10 were thought to reflect "stages of tumour progression." 3•7 Initial three-dimensional analyses suggest that this categorization may need revision. 36 Using principal component analysis, a statistical approach designed to analyse interrelations among a large number of variables and to explain the variables in terms of their common underlying dimensions, Foss
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Microvascular loops and networks-KiveHi et al and associates 20 provided an alternative grouping. Arcs, arcs with branching, normal and silent patterns clustered together and were thought to imply "loss of ordered growth." The cluster of parallel vessels with and without cross-linking was suggested to reflect "orientation of the section." Microvascular loops and networks grouped with straight vessels, and this cluster was thought to imply "fast-growing clones of cells." The two groups agreed that the patterns of normal, arcs, arcs with branching, parallel with cross-linking, loops and networks carry prognostic information, and that loops and networks are most strongly associated with the risk of death from metastasis. 3•20 HISTOLOGIC IDENTIFICATION OF MICROVASCULAR LOOPS AND NETWORKS
According to the original definition of Folberg and colleagues,4 a microvascular loop is a curved pattern that is completely closed, and the presence of one closed loop is sufficient evidence to record this pattern as present. Loops are reminiscent of arcs, defined as curves that are incomplete loops. Networks are defined as at least three back-to-hack closed loops. If networks are present, loops, by definition, are also present. 4 Because networks are formed of loops, for statistical purposes these two patterns can be captured in one ordered three-category variable (no loops, loops without networks, and networks). 17 This convention avoids problems related to multiple comparisons and to analysis of partially interrelated, nested variables. 17 Several staining methods have been used to label microvascular loops and networks and the other microvascular patterns for easy identification. Initially, Folberg and colleagues4 used fluoresceinconjugated Ulex europeaeus agglutinin I, but they found that identical patterns were revealed by PAS, which reacts with periendothelial basement membranes and collagen. Histochemical analysis with azan stain, a modified trichrome stain, also has been used for this purpose, 31 ·32 but azan does not stain exactly the same matrix components as PAS. For efficient identification of microvascular patterns, melanin must be bleached before staining (e.g., with 0.25% potassium permanganate and 5% oxalic acid), no hematoxylin counterstain is applied, and slides are evaluated under a dark green filter (Wratten no. 58, Eastman Kodak, Rochester, NY, or equivalent), which enhances contrast (Fig. 1, Band C). 3 This
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procedure is the "gold standard" when identifying microvascular patterns in malignant tumours. Deviations from it may result in underestimation of microvascular loops and networks (Table 1). If the green filter is omitted, the proportion of tumours with no loops appears to increase by a mean of 15 percentage points (42% vs. 57%), and if bleaching is also omitted and counterstain is applied, the proportion of tumours with no loops appears to increase by a mean of 28 percentage points (42% vs. 71 %) (Table 1). Selection bias does not explain these differences, because all series tabulated were either unselected or rich in mixed and epithelioid cell melanomas, which have loops and networks more often than do spindle cell melanomas. 4·17·46·47 Another important issue is that microvascular loops and networks vary considerably in size and appearance (Fig. 1), more than is evident from the original description. 3·4 Loops typically vary from 14 !Jill to 157 !Jill in diameter and are ovoid in shape. 4 Although individual loops typically are slender to pencil thin (Fig. 1, A and D), some are thicker because of reduplication (Fig. 1, B-G) or associated fibrosis. 2·3 1 A fairly frequent variety is networks of miniature loops, each of which may enclose only a single or a few, often epithelioid, cells (Fig. 1, E and F). When the tumour infiltrates the ciliary body, one must not confuse infiltrated ciliary muscle bundles with microvascular networks (Fig. 1, H)Y When microvascular loops are identified from noncounterstained sections under a green filter, these two can appear deceptively similar. Patterns that are unusually thick and consist of dense fibrovascular tissue may represent ordinary fibrosis, 2 for example after necrosis, rather than microvascular patterns. Investigators feel that loops and networks are somewhat easier to identify than other microvascular patterns, 16 and they can train to identify them consistently enough to achieve good interobserver agreement.3·17 Nevertheless, neophytes are encouraged to familiarize themselves with the full spectrum of microvascular loops and networks using a panel of uveal melanomas so as to learn to grade these patterns correctly. Often, a main source of disagreement is whether a loop is closed or represents a nearly closed arc, and whether a network may in fact be almost closed arcs with branching. 17 For research purposes, we recommend two independent observers, who grade a training set of slides first and resolve any discrepancies by consensus. 3·17
Microvascular loops and networks-KiveHi et al
Table !-Proportion of choroidal and ciliary body melanomas identified as containing microvascular loops and networks according to method used Microvascular pattern; no. (and%) of tumours
Investigator
Evaluated under green filter Folberg et al 3 Makitie et al 17 Seregard et al' 6 Not evaluated under green filter Foss et al 20 Sheidow et al 45 Anastassiou et al 46 McLean et al' 5 Filter not stated Scholes et al 47
Staining modification
Original description* None No bleaching
None None No bleaching No bleaching, counterstain Not stated
No loops
Loops without networks
Networks
103 (44) 54 (40) 56 (44)
26 (II) 33 (25) 48 (38)
105 (45) 47 (35) 24 (19)
63 (54) 28 (61) 59 (66)
25 (21) 7 (IS) 7 (8)
29 (25) II (24) 25 (28)
377 (76)
119 (24)
35 (35)
64 (64)
*"Gold standard": bleaching of melanin with 0.25% potassium permanganate and 5% oxalic acid, periodic acid-Schiff stain without hematoxylin counterstain, and evaluation of staining under a dark green filter (Wratten no. 58, Eastman Kodak, Rochester, NY, or equivalent). 3•4
CLINICAL IDENTIFICATION OF MICROVASCULAR
MICROVASCULAR LOOPS AND NETWORKS IN
LOOPS AND NETWORKS
PRIMARY UVEAL MELANOMA
Microvascular patterns were initially chosen for analysis because they were large enough to be visible by noninvasive methods. Confocal indocyanine green angiography is capable of identifying several patterns in the superficial part of the tumour, as confirmed after enucleation. 48.49 Moreover, with this technique it has been possible both to correlate more rapid reduction of tumour size after brachytherapy to the presence of clinically identified microvascular networks within the tumour, 50 and to identify complex patterns that predict growth of a small melanocytic choroidal tumourY Networks are one such pattern that is relatively straightforward to identify. Another attempt to detect microvascular loops and networks clinically uses acoustic backscatter. 52- 54 Statistical analysis showed that acoustic characteristics were correlated with the presence of matrix-rich patterns in eyes that were later enucleated. 54
Most series of uveal melanomas in which microvascular patterns have been studied were small, selected15·47 or enriched with patients who died from their tumour. 12·46 We characterized a larger populationbased series of 167 choroidal and ciliary body melanomas in eyes enucleated between 1972 and 1981. 17 •22·24·27 ·55 During that period, prompt enucleation was the standard treatment for all but the smallest uveal melanomas, which were first observed for growth. We were able to assess microvascular factors in 80% of the tumours. Closed microvascular loops were detected in 60%, and loops formed networks in 35%. These figures agree with the original report by Folberg and colleagues3 (Table 1), whose series of 234 patients was referral-based. Notably lower proportions of microvascular patterns in choroidal and ciliary body melanomas should arouse suspicion that identification of these patterns is not optimal (Table 1). If the tumours studied are larger
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Fig. 2-Colocalization of microvascular loops and CD34 epitope of endothelial cells in primary ciliochoroidal melanoma. A: A loop (arrowhead) adjacent to arcs with branching and associated macrophages is outlined by CD34-positive channels (B). C: A network with few macrophages corresponds to putative CD34-positive microvascular channels seen in longitudinal and crosssection (D). E: Arcs with adjacent PAS-positive macrophages between bundles of tumour cells mimic networks. F: An area without obvious microvascular patterns contains CD34-positive channels (G), which contain erythrocytes (H). (A, C, E, F: PAS without counterstain; B, D, G: immunoperoxidase; H: hematoxylin-eosin. Magnification: E: x75; C: x 145;A, B, D, F, G, H: x285.)
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than average, like those that are enucleated currently, the proportions should probably be somewhat higher. Only in iris melanomas are loops and networks rare.23,26 Tumour-infiltrating macrophages are a ubiquitous component of untreated primary uveal melanoma. 24•56·57 They are often PAS-positive and are arranged along loops, networks and other microvascular patterns in uveal melanoma (Fig. 2, A and B), 24•31 although such an arrangement is not always found (Fig. 2, C, D and E). In addition, macrophages diffusely infiltrate throughout the tumour. 24 Sometimes, macrophages are arranged around bundles of tumour cells without obvious associated PAS-positive matrix, mimicking loops and networks (Fig. 2, E). Antibodies to vascular endothelial cells, such as those recognizing the CD31 epitope, the CD34 epitope and factor-VIII-related antigen, highlight a population of microvascular channels associated with loops and networks (Fig. 2, B and D). 22 However, the densest areas of microvascular channels are usually located away from these patterns, and even in regions of apparent microvascular "silence" these antibodies identify microvascular spaces containing erythrocytes (Fig. 2, F, G and H). 20·22 It is also possible that melanoma cells acquire immunoreactivity for the CD34 epitope. 25 Although in our experience this is rare, it may contribute to counting of microvascular density. ASSOCIATION WITH OTHER PROGNOSTIC FACTORS
Microvascular loops and networks in untreated primary uveal melanomas are strongly and consistently associated with high microvascular density in hot spots of the tumour20·22 and with the presence of epithelioid cells and cell proliferation (Fig. 3). 4·17·23·46.47 Microvascular density, in tum, is associated with the presence of epithelioid cells and largest basal tumour diameter. 12·22 In spite of these interrelations, loops and networks, microvascular density and epithelioid cells are independently associated with short survival in at least two large series 22·25 and form an important cluster of prognostic factors. Monosomy 3, an important genetic abnormality that is associated with a particularly high risk of metastasis of uveal melanoma, 58-6° is detected in 31% of tumours without loops and in 67% of tumours that have loops, with or without networks. 47 It is currently unclear whether partial deletions may be present in tumours that have networks but no monosomy 3.47·61
Fig. 3-lnterrelations of microvascular loops and networks and other major prognostic factors in uveal melanoma. Thickness of connecting lines corresponds to strength of association, and numbers are p values. MLN = mean of I0 largest nucleoli; MVD = microvascular density; LBD = largest basal tumour diameter. Data from our group 17•22•24•27•55 and Scholes and colleagues.47
Monosomy 3 is also associated with the presence of epithelioid cells (Fig. 3). 47 A less consistent association exists between microvascular loops and networks and involvement of the ciliary body by the tumour,l1· 18·46·47 perhaps because identification of loops and networks in the ciliary body area can be challengingP Loops and networks 47 are only loosely associated with largest basal diameter, a strong clinical indicator of poor prognosis in uveal melanomaP· 22 They are not associated with the mean of the 10 largest nucleoli, a strong indicator of high risk of death from metastasis. 18·27 So far, unconfirmed associations link loops and networks with immunoreactivity for ezrin55 and very late activation antigen 2. 46 Ezrin mediates interaction of actin microfilaments with cell membrane proteins and helps the cell to maintain specialized functions at defined surface environments. Very late activation antigen 2 is an integrin receptor that promotes cell-tocell and cell-to-substratum interactions. Ezrin immunoreactivity in uveal melanoma is more strongly associated with a high number of tumour-infiltrating macrophages and high microvascular density than with loops and networks. 55 These form a second interesting prognostic cluster (Fig. 3).
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Microvascular loops and networks-KiveHi et al The interrelation between macrophages and microvascular patterns is quite complex. Macrophages are often arranged along and within extravascular matrix patterns,24•31 but statistically they are associated with high microvascular density rather than with loops and networks. 24•28 It is not known whether macrophages are causally related to tumour progression and to formation of microvascular patterns (e.g., by secreting growth-promoting cytokines) or whether they are merely scavengers that reflect high cell turnover in rapidly growing, aggressive melanomas. 24 It is postulated that endothelial monocyte-activating protein II, which is found especially around nests of melanoma cells limited by microvascular arcs, loops and networks, attracts macrophages. 32
in which the median duration of follow-up after enucleation was 25 years, 41% of patients without loops, 53% with loops only and 83% with networks died within 10 years after enucleation (Fig. 4, A). 27 Rates of melanoma-related death are 36 to 38 percentage points higher when loops and networks are present than when loops are not found. 3•17 These rates are consistent with those reported by other investigators. 15 •16•20 Loops and networks are particularly useful in differentiating spindle cell melanomas and choroidal melanomas with better and worse prognosisY In our series, increasing microvascular density was strongly associated with death from metastasis (Fig. 4, B). This finding has been reported by most, but not all, investigators. 12•20•25 •45 The results of multivariate analysis vary from study to study, 15 •16•20 and larger series must be accumulated before definite conclusions can be drawn. On proportional hazards regression, microvascular loops and networks (hazard ratio [HR] 1.43 for each category change), presence of epithelioid cells (HR 2.95), microvascular density (HR 1.32 for each unit change
ASSOCIATION WITH MELANOMA-SPECIFIC SURVIVAL
Folberg and colleagues3•4 initially found that the presence of microvascular loops and networks in choroidal melanoma was associated with a high risk of death from metastasis. In our population-based series,
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Fig. 4--Melanoma-specific survival (log-rank test) for 126 patients with primary choroidal and ciliary body melanoma (A, B) and 16 patients with metastatic uveal melanoma (C, D) according to presence of microvascular loops and networks (A, C) and microvascular density (B, D). Ticks indicate censored observations. Microvascular density is counted from "hot spots" and is divided into tertiles. Data from our group. 27•29
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Fig. 5-Difference between primary ciliochoroidal melanoma (A) and its corresponding hepatic metastasis (B), and between irradiated (C) and matched unirradiated (D) primary ciliochoroidal melanoma in presence of microvascular loops and networks (NET) (A, C) and microvascular density counted from hot spots (D). Note that networks tend to be less frequent in irradiated tumours and that microvascular density increases in metastases but is lower in irradiated primary melanomas than in unirradiated primary melanomas. Data from our group. 27•29
in square-root-transformed microvascular density) and mean of the 10 largest nucleoli (HR 1.36 for each micron increase) retained independent statistical significance in our population-based series. 27 In a study by Chen and coworkers, 25 loops, networks and microvascular density likewise were independently associated with prognosis when loops and networks were determined by the gold standard (melanin bleaching and PAS staining without counterstain) and microvascular density was measured from hot spots. TUMOUR PROGRESSION: MICROVASCULAR LOOPS AND NETWORKS IN METASTASES
One way to increase understanding of progression from primary to metastatic melanoma in humans is to compare how tumour characteristics change on dis-
semination. The microcirculation architecture of metastatic choroidal and ciliary body melanoma is not completely understood at present, 21 •29 although Fuchs 1 knew that "alveolar melanomas" produced metastases that were usually alveolar in type. In a nonpaired analysis of 35 metastatic foci that corresponded to 19 primary uveal melanomas, of which 95% contained loops and networks, both microvascular patterns were present in 81% of 10 liver metastases, 88% of 7 lung and skin metastases, and 45% of 11 metastases to other sites. 21 An analysis of 48 uveal melanomas (50% with networks and 67% with loops) with liver metastases showed more variability. 29 A total of 57% of primary melanomas without networks had networks in the corresponding metastasis. On the other hand, 38% of primary melanomas with networks developed
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Microvascular loops and networks-KiveHi et al metastases without networks (Fig. 5, A). Overall, metastases showed loops in 74% of patients and networks in 60% of patients, which are high proportions. The microvascular density of metastases was higher than that of the corresponding primary melanomas (Fig. 5, B). Epithelioid cells were also more frequent in the metastases than in the primary tumours. Networks were present in 17% of the metastases that were 3-4 mm in diameter or smaller in the study by Rummelt and colleagues21 and in 20% of metastases of this size in our study; 29 the proportions were 50% and 92% respectively when the lesion was larger. Since the size and the site of the metastasis were associated in the study by Rummelt and colleagues, one must consider the alternative hypothesis that tumour microenvironment influences the development of microvascular patterns and the growth rate of the metastasis. In our study, however, the effect of metastatic site was eliminated; 29 thus, tumour size may be decisive. It is not known how small a primary melanoma is when networks appear. The smallest primary melanomas with networks in our series were 8 mm in diameter, but networks occur irrespective of tumour height and were often found in primary melanomas that were 1-2 mm thick. It would be interesting to know whether the presence of microvascular loops and networks in metastases is associated with shorter survival after detection of metastases. We provided preliminary evidence that high microvascular density of metastases is associated with shorter overall survival, but we did not have enough biopsied metastases without networks for reliable analysis (Fig. 4, C and D). 29 TUMOUR REGRESSION: MICROVASCULAR LOOPS AND NETWORKS AFTER BRACHYTHERAPY
Irradiation currently is the most common treatment for patients with uveal melanoma, and it is generally believed that irradiation destroys both normal vascular endothelial and tumour cells. However, the behaviour of microvascular loops and networks after irradiation is even less well known than their behaviour on metastasis. 29•50 To formulate a theory as to how irradiation may alter microcirculation attributes, we collected 34 matched pairs of unirradiated and irradiated melanomas in eyes enucleated as primary treatment or after suspected local recurrence and development of treatment complications respectively. 28 Matching was according to
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ciliary body involvement, tumour height at the time of primary treatment, and cell type and grade of pigmentation at the time of enucleation. Loops and networks tended to be found less frequently in the irradiated melanomas than in the unirradiated melanomas. In 13 of 15 pairs in which the unirradiated melanoma had networks, this pattern was not observed in the irradiated melanoma, although 10 of the 13 tumours had loops (Fig. 5, C). In addition, the irradiated melanomas showed more extensive necrosis, and their median microvascular density was 10 counts lower than that of the unirradiated tumours (Fig. 5, D). Microvascular density in the irradiated tumours was not associated with the presence of loops and networks. Our study provides indirect evidence that irradiation reduces microvascular networks and microvascular density, both of which are associated with high risk of metastasis in unirradiated uveal melanomas. This would mean that the risk of dissemination should be smaller even if occasional tumour cells remain capable of cycling after irradiation. Because the irradiated and unirradiated tumours were necessarily from different patients, the possibility remains that brachytherapy may have selected for enucleation less aggressive melanomas that had fewer networks and lower than average microvascular density. If tumours with networks and high microvascular density were more radiosensitive, they might leave a smaller, less leaking and less ischemic residual mass with less chance for recurrence and complications, and the patient might avoid enucleation. Indeed, in a clinical study in which microvascular networks were found by confocal indocyanine green angiography in 11 of 20 uveal melanomas, tumour height regressed by a median of 51% within 1 year of brachytherapy in tumours with networks, compared with 28% in those without networks. 5° The investigators did not determine how networks changed when the tumour regressed. CONCLUSION
The presence of extravascular matrix patterns in uveal melanomas and their association with death from metastasis are well established. Avenues for immediate future research include 1) detailed elucidation of their histogenesis and identification of ways to influence it in order to limit the growth and dissemination of uveal melanoma, and 2) determination of microvascular patterns and density in metastatic melanoma to provide
Microvascular loops and networks-KiveHi et al histopathological characteristics for prognostication when clinical metastases have developed. 29•62 Death from uveal melanoma is a function of several factors. These include the ability of tumour cells, microvessels and microvascular patterns to interact so that neoplastic cells can escape the eye, the ability of tumour cells to grow in the target organs, and the ability of immune surveillance and other host defences to keep tumour cells under control. Microvessels and microvascular patterns may be important in all three regards, because they provide conduits through which macrophages and other effector cells can gain access to primary and metastatic uveal melanomas. Specifically, microvascular loops and networks and microvascular density may reflect parallel processes contributing to metastasis, especially as they are independent predictors of prognosis and are related to different tumour areas. 22•25 They may, for example, differentially reflect the growth rate of the tumour on one hand and the potential of seeding micrometastases on the other. The most malignant tumours would be those that share both microvascular characteristics. Consider the case of an 18-year-old girl from our practice who underwent enucleation for spindle cell choroidal melanoma without microvascular loops. Clinical metastasis developed 20 years later, and the woman died 2 years afterward. The microvascular density of her tumour was in the highest quartile. Did the absence of loops predict low growth potential but, paradoxically, the high microvascular density a high chance of metastasis? We have learned more about the microcirculation of uveal melanoma during the last decade than during the preceding 100 years. The present era of microvascular and genetic research is a fascinating, but as yet unfinished, chapter in the pursuit of a cure for uveal melanoma.
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This work was supported by grants TYH1217 and TYH3203 from the Helsinki University Central Hospital Research Fund, the Finnish Medical Foundation, the Sigrid Juselius Foundation and the Finnish Eye Foundation.
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1. Fuchs E. Das Sarcom des Uvealtractus. Vienna: Wilhelm Braumiiller; 1882. p. 144-7. 2. Folberg R, Mehaffey M, Gardner LM, Meyer M, Rummelt V, Pe'er J. The microcirculation of choroidal and ciliary body melanomas. Eye 1997;1l(Pt 2):227-38. 3. Folberg R, Rummelt V, Parys-van Ginderdeuren R,
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Hwang T, Woolson RF, Pe' er J, et al. The prognostic value of tumor blood vessel morphology in primary uveal melanoma. Ophthalmology 1993;100:1389-98. Folberg R, Pe'er J, Groman LM, Woolson RF, Jeng G, Montague PR, et al. The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: a matched case-control study. Hum Patholl992;23:1298-305. Folberg R, Hendrix MJC, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol2000;156: 361-81. Maniotis AJ, Folberg R, Hess A, Seftor EA, Gardner LM, Pe'er J, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol1999;155:739-52. Rummelt V, Folberg R, Rummelt C, Groman LM, Hwang T, Woolson RF, et al. Microcirculation architecture of melanocytic nevi and malignant melanomas of the ciliary body and choroid. A comparative histopathologic and ultrastructural study. Ophthalmology 1994;101:718-27. Rummelt V, Folberg R, Woolson RF, Hwang T, Pe'er J. Relation between the microcirculation architecture and the aggressive behavior of ciliary body melanomas. Ophthalmology 1995;102:844-51. Mehaffey MG, Gardner LM, Folberg R. Distribution of prognostically important vascular patterns across multiple levels in ciliary body and choroidal melanomas. Am J Ophthalmol1998;126:373-8. Folberg R, Chen X, Boldt HC, Pe'er J, Brown CK, Woolson RF, et al. Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas. Ophthalmology 2001;108:996-1001. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis - correlation in invasive breast carcinoma. N Engl J Med 1991;324:1-8. Foss AJE, Alexander RA, Jefferies LW, Hungerford JL, Harris AL, Lightman S. Microvessel count predicts survival in uveal melanoma. Cancer Res 1996;56:2900-3. Schaling DF, Vander Pol JP, Schlingemann RO, Parysvan Ginderdeuren R, Ruiter DJ, Jager MJ. Vascular density and vascular patterns in the prognosis of choroidal melanoma. In: Schaling DF. Radionuclides and radiolabelled antibodies in choroidal melanoma (diagnosis and therapy) [thesis]. Leiden (The Netherlands): Rijksuniversiteit te Leiden, 1996. Sakamoto T, Sakamoto M, Yoshikawa H, Hata Y, Ishibashi T, Ohnishi Y, et al. Histologic findings and prognosis of uveal malignant melanoma in Japanese patients. Am J Ophthalmol1996;121:276-83. McLean IW, Keefe KS, Burnier MN. Uveal melanoma: comparison of the prognostic value of fibrovascular loops, mean of the ten largest nucleoli, cell type, and tumor size. Ophthalmology 1997;104:777-80. Seregard S, Spangberg B, Juul C, Oskarsson M. Prognostic accuracy of the mean of the largest nucleoli, vascular patterns, and PC-1 0 in posterior uveal melanoma. Ophthalmology 1998;105:485-91.
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17. Makitie T, Summanen P, Tarkkanen A, Kivela T. Microvascular loops and networks as prognostic indicators in choroidal and ciliary body melanomas. J Nat! Cancer Inst 1999;91:359-67. 18. Pe'er J, Rummelt V, Mawn L, Hwang T, Woolson RF, Folberg R. Mean of the ten largest nucleoli, microcirculation architecture, and prognosis of ciliochoroidal melanomas. Ophthalmology 1994;101:1227-35. 19. Daniels KJ, Boldt HC, Martin JA, Gardner LM, Meyer M, Folberg R. Expression of type VI collagen in uveal melanoma: its role in pattern formation and tumor progression. Lab Invest 1996;75:55-66. 20. Foss AJE, Alexander RA, Hungerford JL, Harris AL, Cree IA, Lightman S. Reassessment of the PAS patterns in uveal melanoma. Br J Ophthalmol 1997;81:240-6; discussion 247-8. 21. Rummelt V, Mehaffey MG, Campbell RJ, Pe'er J, Bentler SE, Woolson RF, et al. Microcirculation architecture of metastases from primary ciliary body and choroidal melanomas. Am J Ophthalmol1998;126:303-5. 22. Makitie T, Summanen P, Tarkkanen A, Kivela T. Microvascular density in predicting survival of patients with choroidal and ciliary body melanoma. Invest Ophthalmol Vis Sci 1999;40:2471-80. 23. Chowers I, Folberg R, Livni N, Pe'er J. Comparison of microcirculation patterns and MIB-1 immunoreactivity in iris and posterior uveal melanoma. Ophthalmology 2001; 108:367-71. 24. Makitie T, Summanen P, Tarkkanen A, Kivela T. Tumorinfiltrating macrophages (CD68+ cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci 2001;42: 1414-21. 25. Chen X, Maniatis AJ, Majumdar D, Pe'er J, Folberg R. Uveal melanoma cell staining for CD34 and assessment of tumor vascularity. Invest Ophthalmol Vis Sci 2002;43: 2533-9. 26. Chowers I, Folberg R, Livni N, Pe'er J. p53 immunoreactivity, Ki-67 expression, and microcirculation patterns in melanoma of the iris, ciliary body, and choroid. Curr Eye Res 2002;24: 105-8. 27. Al-Jamal RT, Miikitie T, Kivela T. Nucleolar diameter and microvascular factors as independent predictors of mortality from malignant melanoma of the choroid and ciliary body. Invest Ophthalmol Vis Sci 2003;44:2381-9. 28. Toivonen P, Miikitie T, Kujala E, Kivela T. Macrophages and microcirculation in regressed and partially regressed irradiated choroidal and ciliary body melanomas. Curr Eye Res 2003;27:237-45. 29. Toivonen P, Miikitie T, Kujala E, Kivela T. Microcirculation and tumor-infiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases. Invest Ophthalmol Vis Sci 2004;45:1-6. 30. Folberg R, Fleck M, Mehaffey MG, Meyer M, Bentler SE, Woolson RF, et al. Mapping the location of prognostically significant microcirculatory patterns in ciliary body and choroidal melanomas. Pathol Oneal Res 1996; 2:229-36.
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31. Clarijs R, Otte-Holler I, Ruiter DJ, de Waal RM. Presence of a fluid-conducting meshwork in xenografted cutaneous and primary human uveal melanoma. Invest Ophthalmol Vis Sci 2002;43:912-8. 32. Clarijs R, Schalkwijk L, Ruiter DJ, de Waal RM. EMAPII expression is associated with macrophage accumulation in primary uveal melanoma. Invest Ophthalmol Vis Sci 2003;44:1801-6. 33. Bissell MJ. Tumor plasticity allows vasculogenic mimicry, a novel form of angiogenic switch -A rose by any other name? Am J Pathol2000;155:675-9. 34. McDonald DM, Munn L, Jain RK. Vasculogenic mimicry: How convincing, how novel, and how significant? Am J Pathol2000;156:383-8. 35. McDonald DM, Foss AJE. Endothelial cells of tumor vessels: abnormal but not absent. Cancer Metastasis Rev 2000;19:109-20. 36. Chen X, Ai Z, Rasmussen M, Bajcsy P, Auvil L, Welge M, et al. Three-dimensional reconstruction of extravascular matrix patterns and blood vessels in human uveal melanoma tissue: techniques and preliminary findings. Invest Ophthalmol Vis Sci 2003;44:2834-40. 37. Warso MA, Maniotis AJ, Chen X, Majumdar D, Patel MK, Shilkaitis A, et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary cutaneous melanoma. Clin Cancer Res 2001;7:473-7. 38. Thies A, Mangold U, Moll I, Schumacher U. PAS-positive loops and networks as a prognostic indicator in cutaneous malignant melanoma. J Pathol2001;195:537-42. 39. Lee YJ, Nagai N, Siar CH, Nakano K, Nagatsuka H, Tsujigiwa H, et al. Angioarchitecture of primary oral malignant melanomas. J Histochem Cytochem 2002;50:1555-62. 40. Buijs JT, Cleton AM, Smit VT, Lowik CW, E Papapoulos S, Pluijm G. Prognostic significance of periodic acidSchiff-positive patterns in primary breast cancer and its lymph node metastases. Breast Cancer Res Treat 2004; 84:117-30. 41. Sood AK, Seftor EA, Fletcher MS, Gardner LM, Heidger PM, Buller RE, et al. Molecular determinants of ovarian cancer plasticity. Am J Pathol2001;158:1279-88. 42. SharmaN, Seftor RE, Seftor EA, Groman LM, Heidger PM Jr, Cohen MB, et al. Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry. Prostate 2002;50: 189-201. 43. Maniotis AJ, Chen X, Garcia C, DeChristopher PJ, Wu D, Pe'er J, et al. Control of melanoma morphogenesis, endothelial survival, and perfusion by extracellular matrix. Lab Invest 2002;82:1031-43. 44. Kivela T, Eskelin S, Miikitie T, Summanen P. Exudative retinal detachment from malignant uveal melanoma: predictors and prognostic significance. Invest Ophthalmol Vis Sci 2001;42:2085-93. 45. Sheidow TG, Hooper PL, Crukley C, Young J, Heathcote JG. Expression of vascular endothelial growth factor in uveal melanoma and its correlation with metastasis. Br J Ophthalmol2000;84:750--6.
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46. Anastassiou G, Schilling H, Djakovic S, Bornfeld N. Expression of VLA-2, VLA-3, and alpha(v) integrin receptors in uveal melanoma: association with microvascular architecture of the tumour and prognostic value. Br J Ophthalmol2000;84:899-902. 47. Scholes AG, Damato BE, Nunn J, Hiscott P, Grierson I, Field JK. Monosomy 3 in uveal melanoma: correlation with clinical and histologic predictors of survival. Invest Ophthalmol Vis Sci 2003;44:1008-11. 48. Mueller AJ, Bartsch DU, Folberg R, Mehaffey MG, Boldt HC, Meyer M, et al. Imaging the microvasculature of choroidal melanomas with confocal indocyanine green scanning laser ophthalmoscopy. Arch Ophthalmol 1998; 116:31-9. 49. Mueller AJ, Freeman WR, Folberg R, Bartsch DU, Scheider A, Schaller U, et al. Evaluation of microvascularization pattern visibility in human choroidal melanomas: comparison of confocal fluorescein with indocyanine green angiography. Graefes Arch Clin Exp Ophthalmol 1999;237:448-56. 50. Schaller UC, Mueller AJ, Bartsch DU, Schaumberger M, Freeman WR, Kampik A. Zusarnmenhang von ICGangiographisch nachgewiesenen "Networks" uvealer Melanome und Tumorriickbildungsgeschwindigkeit nach Brachytherapie. Ophthalmologe 2002;99:545-8. 51. Mueller AJ, Freeman WR, Schaller UC, Kampik A, Folberg R. Complex microcirculation patterns detected by confocal indocyanine green angiography predict time to growth of small choroidal melanocytic tumors: MuSIC report II. Ophthalmology 2002;109:2207-14. 52. Coleman DJ, Rondeau MJ, Silverman RH, Folberg R, Rummelt V, Woods SM, et al. Correlation of microcirculation architecture with ultrasound backscatter parameters of uveal melanoma. Eur J Ophthalmol1995;5:96-106. 53. Silverman RH, Folberg R, Boldt HC, Lloyd HO, Rondeau MJ, Mehaffey MG, et al. Correlation of ultrasound parameter imaging with microcirculatory patterns in
uveal melanomas. Ultrasound Med Biol1997;23:573-81. 54. Silverman RH, Folberg R, Rondeau MJ, Boldt HC, Lloyd HO, Chen X, et al. Spectral parameter imaging for detection of prognostically significant histologic features in uveal melanoma. Ultrasound Med Biol2003;29:951-9. 55. Miikitie T, Carpen 0, Vaheri A, Kivela T. Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma. Invest Ophthalmol Vis Sci 2001;42:2442-9. 56. Tobal K, Deuble K, McCartney A, Lightrnan S. Characterization of cellular infiltration in choroidal melanoma. Melanoma Res 1993;3:63-5. 57. Collaborative Ocular Melanoma Study Group. Histopathologic characteristics of uveal melanomas in eyes enucleated from the Collaborative Ocular Melanoma Study. COMS report no. 6. Am J Ophthalmoll998;125:745-66. 58. Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jackel KH, Becher R. Prognostic implications of monosomy 3 in uveal melanoma. Lancet 1996;347:1222-5. 59. Sisley K, Rennie IG, Parsons MA, Jacques R, Hammond DW, Bell SM, et al. Abnormalities of chromosomes 3 and 8 in posterior uveal melanoma correlate with prognosis. Genes Chromosomes Cancer 1997;19:22-8. 60. White VA, Chambers JD, Courtright PD, Chang WY, Horsman DE. Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer 1998;83:354-9. 61. Parrella P, Fazio VM, Gallo AP, Sidransky D, Merbs SL. Fine mapping of chromosome 3 in uveal melanoma: identification of a minimal region of deletion on chromosomal arm 3p25.1-p25.2. Cancer Res 2003;63:8507-10. 62. Eskelin S, PyrhOnen S, Hahka-Kemppinen M, Tuomaala S, Kivela T. A prognostic model and staging for metastatic uveal melanoma. Cancer 2003;97:465-75.
Key words: macrophages, metastasis, microvessels, survival, uveal melanoma
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n his 1882 monograph Das Sarcom des Uvealtractus, Fuchs 1 drew attention to a subgroup of uveal melanomas that his contemporaries called "alveolar sarcoma" and "combined sarcoma and carcinoma." He identified 14 tumours of this type among 258 uveal melanomas known to him and noted that they were particularly rapidly growing, so that enucleation became necessary an average of 10 months after the
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From the Ocular Oncology Service and Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland Correspondence to: Dr. Tero Kivela, Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, Haartrnaninkatu 4 C, PL 220, FIN-00029 HUS, Helsinki, Finland; tero.k:[email protected] Can J Ophthalmol 2004;39:409-21
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onset of symptoms, 21 months earlier than usual. Fuchs thought that these were the most malignant uveal melanomas, because 8 of the 14 patients had died from metastasis. 1 He found that histologically the metastases repeated the alveolar structure of the primary uveal tumour. In retrospect, some of the tumours that Fuchs observed may have appeared alveolar because of microvascular networks, 2-6 a matrix pattern found in uveal melanomas that occasionally is very conspicuous even on routine light microscopy (Fig. 1, A). Fuchs did not specifically link the pattern with tumour vascularization, far less discover that networks are but a special case of a novel type of tumour microcirculation that is now known as vasculogenic mimicry. 5•6 This discovery was left for other investigators to make. In 1992 Folberg and colleagues4 searched for light microscopic features of uveal melanoma that would
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Fig. !-Spectrum of microvascular networks in primary ciliochoroidal melanoma (A, D, E, F, H) and its metastases (B, C). A: Microvascular networks in amelanotic choroidal melanoma on routine hematoxylin-eosin staining. Note erythrocytes between matrix layers (arrowheads). B: Networks are better visualized after "gold standard" melanin bleaching and periodic acid-Schiff reagent (PAS) staining without counterstain. C: Identification is further aided by using dark green filter. 0: Pencil-thin networks and associated PAS-positive macrophages (arrowheads) in spindle cell melanoma. Reduplicated matrix forms networks of miniature loops (E) that enclose single epithelioid cells (arrowheads) (F). G: Thicker networks with macrophages in liver metastasis. H: Ciliary muscle bundles mimic microvascular networks. (A, F: hematoxylin-eosin; B, C, D, E, G, H: PAS without counterstain. Magnification: B, C, G: x75; A, D: xl45; E, F, H: x285.)
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Microvascular loops and networks-KiveHi et a1 be associated with survival and large enough to be visualized noninvasively. They noted that the arrangement of microvessels varied from tumour to tumour and that these patterns fell into nine categories, one of which was microvascular networks. Certain patterns were typical of nevi and low-grade uveal melanomas, 7 whereas· others predicted a high risk of metastasis after enucleation. 3.4.s-10 These investigators were the first to understand the potential implications of this observation, and so commenced the era of microcirculation in the study of uveal melanoma. A related line of research concentrated on microvascular density, which in 1991 had been linked with the risk of metastasis in breast cancer. 11 In 1996 Foss and associates 12 confirmed that high numbers of immunohistochemically labelled microvessels in areas of densest vascularization, or "hot spots," were associated with death after enucleation of uveal melanoma. Although initially challenged, the observations of both groups were later confirmed beyond reasonable doubt.l 3- 17 Although the topic of our review is microvascular loops and networks, we find it important to emphasize that we concur with Folberg and colleagues, 10 who stressed that other matrix patterns, in particular parallel with cross-linking, arcs and arcs with branching, are also associated with risk of metastasis. We focus on loops and networks because they have been most strongly and consistently associated with risk of death from metastasis of uveal melanoma and because these two are by far the most extensively studied of the nine microvascular patterns. 2-4,6-10,I5-29
These networks may provide a novel form of circulation to malignant tumours in addition to classic angiogenesis and vascular cooption. 5 Endothelial-cell-lined blood vessels can be embedded between the matrix sheets. 31 •36 Vasculogenic mimicry has subsequently been described in other cancers, such as cutaneous melanoma6•37- 39 and breast, 40 ovarian41 and prostate42 carcinoma. It has been difficult to prove conclusively the presence in human uveal melanoma of microvascular channels, which are not lined by endothelial cells, 35 although electron microscopic images have suggested that they exist. 5•6•43 In three-dimensional cultures, aggressive uveal melanoma cells capable of metastasis form PAS-positive looping and networking channels, which consist of laminin and other basement membrane components. Tracer substance can be injected through the channels.6.43 Likewise, in xenografts of cutaneous melanoma, sheets of extracellular matrix rich in laminin and collagen are deposited around clusters of tumour cells. 31 Slits between the sheets seem to conduct fluid, and they may functionally mimic lymphatic vessels. These spaces also contain macrophages. 31 These are interesting observations, because the presence of loops and networks in uveal melanoma increases the likelihood of exudative retinal detachment. 44 A melanoma that has networks is four times as likely to cause retinal detachment as a melanoma of the same size and location but without loops and networks. 44 Interstitial fluid conducted through extravascular networks may explain this association. HIERARCHY OF MICROVASCULAR PATTERNS
fORMATION OF MICROVASCULAR PATTERNS
Different opinions about the histogenesis, nature and function of the potentially perfused connective tissue patterns, which can be identified with periodic acid-Schiff stain (PAS), have led to variable terminology over time: they have been called vascular, 4 fibrovascular,l5 PAS, 20 microcirculatory30 and, most recently, extravascular matrix 31 •32 patterns. A key hypothesis, which has aroused great interest and debate, 33-35 maintains that uveal melanomas can acquire part of their circulation by generating patterned extravascular matrix channels that conduct plasma and, sometimes, blood. 5•6 The formation of these threedimensional, matrix-rich, vascular-like networks together with deregulated expression of genes typical of vascular cells is designated vasculogenic mimicry. 5•6
According to Folberg and colleagues, 10 the nine microvascular patterns form two groups: those of linear patterns ("straight," "parallel" and "parallel with cross-linking") and those of curved patterns ("arcs," "arcs with branching," "loops" and "networks"). The patterns of "silence" and "normal vessels" appear to belong to a third group that is characteristic of uveal nevi. 7 These three groups, which are interrelated, 10 were thought to reflect "stages of tumour progression." 3•7 Initial three-dimensional analyses suggest that this categorization may need revision. 36 Using principal component analysis, a statistical approach designed to analyse interrelations among a large number of variables and to explain the variables in terms of their common underlying dimensions, Foss
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Microvascular loops and networks-KiveHi et al and associates 20 provided an alternative grouping. Arcs, arcs with branching, normal and silent patterns clustered together and were thought to imply "loss of ordered growth." The cluster of parallel vessels with and without cross-linking was suggested to reflect "orientation of the section." Microvascular loops and networks grouped with straight vessels, and this cluster was thought to imply "fast-growing clones of cells." The two groups agreed that the patterns of normal, arcs, arcs with branching, parallel with cross-linking, loops and networks carry prognostic information, and that loops and networks are most strongly associated with the risk of death from metastasis. 3•20 HISTOLOGIC IDENTIFICATION OF MICROVASCULAR LOOPS AND NETWORKS
According to the original definition of Folberg and colleagues,4 a microvascular loop is a curved pattern that is completely closed, and the presence of one closed loop is sufficient evidence to record this pattern as present. Loops are reminiscent of arcs, defined as curves that are incomplete loops. Networks are defined as at least three back-to-hack closed loops. If networks are present, loops, by definition, are also present. 4 Because networks are formed of loops, for statistical purposes these two patterns can be captured in one ordered three-category variable (no loops, loops without networks, and networks). 17 This convention avoids problems related to multiple comparisons and to analysis of partially interrelated, nested variables. 17 Several staining methods have been used to label microvascular loops and networks and the other microvascular patterns for easy identification. Initially, Folberg and colleagues4 used fluoresceinconjugated Ulex europeaeus agglutinin I, but they found that identical patterns were revealed by PAS, which reacts with periendothelial basement membranes and collagen. Histochemical analysis with azan stain, a modified trichrome stain, also has been used for this purpose, 31 ·32 but azan does not stain exactly the same matrix components as PAS. For efficient identification of microvascular patterns, melanin must be bleached before staining (e.g., with 0.25% potassium permanganate and 5% oxalic acid), no hematoxylin counterstain is applied, and slides are evaluated under a dark green filter (Wratten no. 58, Eastman Kodak, Rochester, NY, or equivalent), which enhances contrast (Fig. 1, Band C). 3 This
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procedure is the "gold standard" when identifying microvascular patterns in malignant tumours. Deviations from it may result in underestimation of microvascular loops and networks (Table 1). If the green filter is omitted, the proportion of tumours with no loops appears to increase by a mean of 15 percentage points (42% vs. 57%), and if bleaching is also omitted and counterstain is applied, the proportion of tumours with no loops appears to increase by a mean of 28 percentage points (42% vs. 71 %) (Table 1). Selection bias does not explain these differences, because all series tabulated were either unselected or rich in mixed and epithelioid cell melanomas, which have loops and networks more often than do spindle cell melanomas. 4·17·46·47 Another important issue is that microvascular loops and networks vary considerably in size and appearance (Fig. 1), more than is evident from the original description. 3·4 Loops typically vary from 14 !Jill to 157 !Jill in diameter and are ovoid in shape. 4 Although individual loops typically are slender to pencil thin (Fig. 1, A and D), some are thicker because of reduplication (Fig. 1, B-G) or associated fibrosis. 2·3 1 A fairly frequent variety is networks of miniature loops, each of which may enclose only a single or a few, often epithelioid, cells (Fig. 1, E and F). When the tumour infiltrates the ciliary body, one must not confuse infiltrated ciliary muscle bundles with microvascular networks (Fig. 1, H)Y When microvascular loops are identified from noncounterstained sections under a green filter, these two can appear deceptively similar. Patterns that are unusually thick and consist of dense fibrovascular tissue may represent ordinary fibrosis, 2 for example after necrosis, rather than microvascular patterns. Investigators feel that loops and networks are somewhat easier to identify than other microvascular patterns, 16 and they can train to identify them consistently enough to achieve good interobserver agreement.3·17 Nevertheless, neophytes are encouraged to familiarize themselves with the full spectrum of microvascular loops and networks using a panel of uveal melanomas so as to learn to grade these patterns correctly. Often, a main source of disagreement is whether a loop is closed or represents a nearly closed arc, and whether a network may in fact be almost closed arcs with branching. 17 For research purposes, we recommend two independent observers, who grade a training set of slides first and resolve any discrepancies by consensus. 3·17
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Table !-Proportion of choroidal and ciliary body melanomas identified as containing microvascular loops and networks according to method used Microvascular pattern; no. (and%) of tumours
Investigator
Evaluated under green filter Folberg et al 3 Makitie et al 17 Seregard et al' 6 Not evaluated under green filter Foss et al 20 Sheidow et al 45 Anastassiou et al 46 McLean et al' 5 Filter not stated Scholes et al 47
Staining modification
Original description* None No bleaching
None None No bleaching No bleaching, counterstain Not stated
No loops
Loops without networks
Networks
103 (44) 54 (40) 56 (44)
26 (II) 33 (25) 48 (38)
105 (45) 47 (35) 24 (19)
63 (54) 28 (61) 59 (66)
25 (21) 7 (IS) 7 (8)
29 (25) II (24) 25 (28)
377 (76)
119 (24)
35 (35)
64 (64)
*"Gold standard": bleaching of melanin with 0.25% potassium permanganate and 5% oxalic acid, periodic acid-Schiff stain without hematoxylin counterstain, and evaluation of staining under a dark green filter (Wratten no. 58, Eastman Kodak, Rochester, NY, or equivalent). 3•4
CLINICAL IDENTIFICATION OF MICROVASCULAR
MICROVASCULAR LOOPS AND NETWORKS IN
LOOPS AND NETWORKS
PRIMARY UVEAL MELANOMA
Microvascular patterns were initially chosen for analysis because they were large enough to be visible by noninvasive methods. Confocal indocyanine green angiography is capable of identifying several patterns in the superficial part of the tumour, as confirmed after enucleation. 48.49 Moreover, with this technique it has been possible both to correlate more rapid reduction of tumour size after brachytherapy to the presence of clinically identified microvascular networks within the tumour, 50 and to identify complex patterns that predict growth of a small melanocytic choroidal tumourY Networks are one such pattern that is relatively straightforward to identify. Another attempt to detect microvascular loops and networks clinically uses acoustic backscatter. 52- 54 Statistical analysis showed that acoustic characteristics were correlated with the presence of matrix-rich patterns in eyes that were later enucleated. 54
Most series of uveal melanomas in which microvascular patterns have been studied were small, selected15·47 or enriched with patients who died from their tumour. 12·46 We characterized a larger populationbased series of 167 choroidal and ciliary body melanomas in eyes enucleated between 1972 and 1981. 17 •22·24·27 ·55 During that period, prompt enucleation was the standard treatment for all but the smallest uveal melanomas, which were first observed for growth. We were able to assess microvascular factors in 80% of the tumours. Closed microvascular loops were detected in 60%, and loops formed networks in 35%. These figures agree with the original report by Folberg and colleagues3 (Table 1), whose series of 234 patients was referral-based. Notably lower proportions of microvascular patterns in choroidal and ciliary body melanomas should arouse suspicion that identification of these patterns is not optimal (Table 1). If the tumours studied are larger
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Fig. 2-Colocalization of microvascular loops and CD34 epitope of endothelial cells in primary ciliochoroidal melanoma. A: A loop (arrowhead) adjacent to arcs with branching and associated macrophages is outlined by CD34-positive channels (B). C: A network with few macrophages corresponds to putative CD34-positive microvascular channels seen in longitudinal and crosssection (D). E: Arcs with adjacent PAS-positive macrophages between bundles of tumour cells mimic networks. F: An area without obvious microvascular patterns contains CD34-positive channels (G), which contain erythrocytes (H). (A, C, E, F: PAS without counterstain; B, D, G: immunoperoxidase; H: hematoxylin-eosin. Magnification: E: x75; C: x 145;A, B, D, F, G, H: x285.)
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than average, like those that are enucleated currently, the proportions should probably be somewhat higher. Only in iris melanomas are loops and networks rare.23,26 Tumour-infiltrating macrophages are a ubiquitous component of untreated primary uveal melanoma. 24•56·57 They are often PAS-positive and are arranged along loops, networks and other microvascular patterns in uveal melanoma (Fig. 2, A and B), 24•31 although such an arrangement is not always found (Fig. 2, C, D and E). In addition, macrophages diffusely infiltrate throughout the tumour. 24 Sometimes, macrophages are arranged around bundles of tumour cells without obvious associated PAS-positive matrix, mimicking loops and networks (Fig. 2, E). Antibodies to vascular endothelial cells, such as those recognizing the CD31 epitope, the CD34 epitope and factor-VIII-related antigen, highlight a population of microvascular channels associated with loops and networks (Fig. 2, B and D). 22 However, the densest areas of microvascular channels are usually located away from these patterns, and even in regions of apparent microvascular "silence" these antibodies identify microvascular spaces containing erythrocytes (Fig. 2, F, G and H). 20·22 It is also possible that melanoma cells acquire immunoreactivity for the CD34 epitope. 25 Although in our experience this is rare, it may contribute to counting of microvascular density. ASSOCIATION WITH OTHER PROGNOSTIC FACTORS
Microvascular loops and networks in untreated primary uveal melanomas are strongly and consistently associated with high microvascular density in hot spots of the tumour20·22 and with the presence of epithelioid cells and cell proliferation (Fig. 3). 4·17·23·46.47 Microvascular density, in tum, is associated with the presence of epithelioid cells and largest basal tumour diameter. 12·22 In spite of these interrelations, loops and networks, microvascular density and epithelioid cells are independently associated with short survival in at least two large series 22·25 and form an important cluster of prognostic factors. Monosomy 3, an important genetic abnormality that is associated with a particularly high risk of metastasis of uveal melanoma, 58-6° is detected in 31% of tumours without loops and in 67% of tumours that have loops, with or without networks. 47 It is currently unclear whether partial deletions may be present in tumours that have networks but no monosomy 3.47·61
Fig. 3-lnterrelations of microvascular loops and networks and other major prognostic factors in uveal melanoma. Thickness of connecting lines corresponds to strength of association, and numbers are p values. MLN = mean of I0 largest nucleoli; MVD = microvascular density; LBD = largest basal tumour diameter. Data from our group 17•22•24•27•55 and Scholes and colleagues.47
Monosomy 3 is also associated with the presence of epithelioid cells (Fig. 3). 47 A less consistent association exists between microvascular loops and networks and involvement of the ciliary body by the tumour,l1· 18·46·47 perhaps because identification of loops and networks in the ciliary body area can be challengingP Loops and networks 47 are only loosely associated with largest basal diameter, a strong clinical indicator of poor prognosis in uveal melanomaP· 22 They are not associated with the mean of the 10 largest nucleoli, a strong indicator of high risk of death from metastasis. 18·27 So far, unconfirmed associations link loops and networks with immunoreactivity for ezrin55 and very late activation antigen 2. 46 Ezrin mediates interaction of actin microfilaments with cell membrane proteins and helps the cell to maintain specialized functions at defined surface environments. Very late activation antigen 2 is an integrin receptor that promotes cell-tocell and cell-to-substratum interactions. Ezrin immunoreactivity in uveal melanoma is more strongly associated with a high number of tumour-infiltrating macrophages and high microvascular density than with loops and networks. 55 These form a second interesting prognostic cluster (Fig. 3).
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Microvascular loops and networks-KiveHi et al The interrelation between macrophages and microvascular patterns is quite complex. Macrophages are often arranged along and within extravascular matrix patterns,24•31 but statistically they are associated with high microvascular density rather than with loops and networks. 24•28 It is not known whether macrophages are causally related to tumour progression and to formation of microvascular patterns (e.g., by secreting growth-promoting cytokines) or whether they are merely scavengers that reflect high cell turnover in rapidly growing, aggressive melanomas. 24 It is postulated that endothelial monocyte-activating protein II, which is found especially around nests of melanoma cells limited by microvascular arcs, loops and networks, attracts macrophages. 32
in which the median duration of follow-up after enucleation was 25 years, 41% of patients without loops, 53% with loops only and 83% with networks died within 10 years after enucleation (Fig. 4, A). 27 Rates of melanoma-related death are 36 to 38 percentage points higher when loops and networks are present than when loops are not found. 3•17 These rates are consistent with those reported by other investigators. 15 •16•20 Loops and networks are particularly useful in differentiating spindle cell melanomas and choroidal melanomas with better and worse prognosisY In our series, increasing microvascular density was strongly associated with death from metastasis (Fig. 4, B). This finding has been reported by most, but not all, investigators. 12•20•25 •45 The results of multivariate analysis vary from study to study, 15 •16•20 and larger series must be accumulated before definite conclusions can be drawn. On proportional hazards regression, microvascular loops and networks (hazard ratio [HR] 1.43 for each category change), presence of epithelioid cells (HR 2.95), microvascular density (HR 1.32 for each unit change
ASSOCIATION WITH MELANOMA-SPECIFIC SURVIVAL
Folberg and colleagues3•4 initially found that the presence of microvascular loops and networks in choroidal melanoma was associated with a high risk of death from metastasis. In our population-based series,
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Fig. 4--Melanoma-specific survival (log-rank test) for 126 patients with primary choroidal and ciliary body melanoma (A, B) and 16 patients with metastatic uveal melanoma (C, D) according to presence of microvascular loops and networks (A, C) and microvascular density (B, D). Ticks indicate censored observations. Microvascular density is counted from "hot spots" and is divided into tertiles. Data from our group. 27•29
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in square-root-transformed microvascular density) and mean of the 10 largest nucleoli (HR 1.36 for each micron increase) retained independent statistical significance in our population-based series. 27 In a study by Chen and coworkers, 25 loops, networks and microvascular density likewise were independently associated with prognosis when loops and networks were determined by the gold standard (melanin bleaching and PAS staining without counterstain) and microvascular density was measured from hot spots. TUMOUR PROGRESSION: MICROVASCULAR LOOPS AND NETWORKS IN METASTASES
One way to increase understanding of progression from primary to metastatic melanoma in humans is to compare how tumour characteristics change on dis-
semination. The microcirculation architecture of metastatic choroidal and ciliary body melanoma is not completely understood at present, 21 •29 although Fuchs 1 knew that "alveolar melanomas" produced metastases that were usually alveolar in type. In a nonpaired analysis of 35 metastatic foci that corresponded to 19 primary uveal melanomas, of which 95% contained loops and networks, both microvascular patterns were present in 81% of 10 liver metastases, 88% of 7 lung and skin metastases, and 45% of 11 metastases to other sites. 21 An analysis of 48 uveal melanomas (50% with networks and 67% with loops) with liver metastases showed more variability. 29 A total of 57% of primary melanomas without networks had networks in the corresponding metastasis. On the other hand, 38% of primary melanomas with networks developed
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Microvascular loops and networks-KiveHi et al metastases without networks (Fig. 5, A). Overall, metastases showed loops in 74% of patients and networks in 60% of patients, which are high proportions. The microvascular density of metastases was higher than that of the corresponding primary melanomas (Fig. 5, B). Epithelioid cells were also more frequent in the metastases than in the primary tumours. Networks were present in 17% of the metastases that were 3-4 mm in diameter or smaller in the study by Rummelt and colleagues21 and in 20% of metastases of this size in our study; 29 the proportions were 50% and 92% respectively when the lesion was larger. Since the size and the site of the metastasis were associated in the study by Rummelt and colleagues, one must consider the alternative hypothesis that tumour microenvironment influences the development of microvascular patterns and the growth rate of the metastasis. In our study, however, the effect of metastatic site was eliminated; 29 thus, tumour size may be decisive. It is not known how small a primary melanoma is when networks appear. The smallest primary melanomas with networks in our series were 8 mm in diameter, but networks occur irrespective of tumour height and were often found in primary melanomas that were 1-2 mm thick. It would be interesting to know whether the presence of microvascular loops and networks in metastases is associated with shorter survival after detection of metastases. We provided preliminary evidence that high microvascular density of metastases is associated with shorter overall survival, but we did not have enough biopsied metastases without networks for reliable analysis (Fig. 4, C and D). 29 TUMOUR REGRESSION: MICROVASCULAR LOOPS AND NETWORKS AFTER BRACHYTHERAPY
Irradiation currently is the most common treatment for patients with uveal melanoma, and it is generally believed that irradiation destroys both normal vascular endothelial and tumour cells. However, the behaviour of microvascular loops and networks after irradiation is even less well known than their behaviour on metastasis. 29•50 To formulate a theory as to how irradiation may alter microcirculation attributes, we collected 34 matched pairs of unirradiated and irradiated melanomas in eyes enucleated as primary treatment or after suspected local recurrence and development of treatment complications respectively. 28 Matching was according to
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ciliary body involvement, tumour height at the time of primary treatment, and cell type and grade of pigmentation at the time of enucleation. Loops and networks tended to be found less frequently in the irradiated melanomas than in the unirradiated melanomas. In 13 of 15 pairs in which the unirradiated melanoma had networks, this pattern was not observed in the irradiated melanoma, although 10 of the 13 tumours had loops (Fig. 5, C). In addition, the irradiated melanomas showed more extensive necrosis, and their median microvascular density was 10 counts lower than that of the unirradiated tumours (Fig. 5, D). Microvascular density in the irradiated tumours was not associated with the presence of loops and networks. Our study provides indirect evidence that irradiation reduces microvascular networks and microvascular density, both of which are associated with high risk of metastasis in unirradiated uveal melanomas. This would mean that the risk of dissemination should be smaller even if occasional tumour cells remain capable of cycling after irradiation. Because the irradiated and unirradiated tumours were necessarily from different patients, the possibility remains that brachytherapy may have selected for enucleation less aggressive melanomas that had fewer networks and lower than average microvascular density. If tumours with networks and high microvascular density were more radiosensitive, they might leave a smaller, less leaking and less ischemic residual mass with less chance for recurrence and complications, and the patient might avoid enucleation. Indeed, in a clinical study in which microvascular networks were found by confocal indocyanine green angiography in 11 of 20 uveal melanomas, tumour height regressed by a median of 51% within 1 year of brachytherapy in tumours with networks, compared with 28% in those without networks. 5° The investigators did not determine how networks changed when the tumour regressed. CONCLUSION
The presence of extravascular matrix patterns in uveal melanomas and their association with death from metastasis are well established. Avenues for immediate future research include 1) detailed elucidation of their histogenesis and identification of ways to influence it in order to limit the growth and dissemination of uveal melanoma, and 2) determination of microvascular patterns and density in metastatic melanoma to provide
Microvascular loops and networks-KiveHi et al histopathological characteristics for prognostication when clinical metastases have developed. 29•62 Death from uveal melanoma is a function of several factors. These include the ability of tumour cells, microvessels and microvascular patterns to interact so that neoplastic cells can escape the eye, the ability of tumour cells to grow in the target organs, and the ability of immune surveillance and other host defences to keep tumour cells under control. Microvessels and microvascular patterns may be important in all three regards, because they provide conduits through which macrophages and other effector cells can gain access to primary and metastatic uveal melanomas. Specifically, microvascular loops and networks and microvascular density may reflect parallel processes contributing to metastasis, especially as they are independent predictors of prognosis and are related to different tumour areas. 22•25 They may, for example, differentially reflect the growth rate of the tumour on one hand and the potential of seeding micrometastases on the other. The most malignant tumours would be those that share both microvascular characteristics. Consider the case of an 18-year-old girl from our practice who underwent enucleation for spindle cell choroidal melanoma without microvascular loops. Clinical metastasis developed 20 years later, and the woman died 2 years afterward. The microvascular density of her tumour was in the highest quartile. Did the absence of loops predict low growth potential but, paradoxically, the high microvascular density a high chance of metastasis? We have learned more about the microcirculation of uveal melanoma during the last decade than during the preceding 100 years. The present era of microvascular and genetic research is a fascinating, but as yet unfinished, chapter in the pursuit of a cure for uveal melanoma.
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This work was supported by grants TYH1217 and TYH3203 from the Helsinki University Central Hospital Research Fund, the Finnish Medical Foundation, the Sigrid Juselius Foundation and the Finnish Eye Foundation.
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Key words: macrophages, metastasis, microvessels, survival, uveal melanoma
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