Affiliation of authors: Department of Ophthalmology,Helsinki University Central Hospital, Finland.
Correspondence to: Tero Kivelä, M.D., Vitreoretinal and Oncology Service, Department of Ophthalmology, Helsinki University Central Hospital, Haartmaninkatu 4 C, P.O. Box 220, FIN-00029 HYKS, Finland (e-mail: tero.kivela{at}helsinki.fi).
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ABSTRACT |
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INTRODUCTION |
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Recently, Folberg et al. (5,6) addressed the possibility that the arrangement of microcirculation in uveal melanoma might reflect tumor progression and predict the likelihood of dissemination. They described nine microvascular patterns by histology (5) and showed by multivariate analysis that some of them were better prognostic indicators than conventional clinical or histopathologic characteristics (7), including the size of the tumor (5,8), involvement of the ciliary body (5,9), the cell type (5,8), and the mean size of the 10 largest nucleoli (10).
Subsequent research has identified hierarchical groups among the vascular patterns (11-13). According to Folberg et al. (6), the patterns straight vessels, parallel straight vessels, and parallel straight vessels with cross-linking cluster together, as do arcs, arcs with branching, closed vascular loops, and networks. The normal and silent patterns form a third cluster, which were thought to reflect "stages of tumor progression" (6). In principal component analysis by Foss et al. (13), arcs, arcs with branching, normal pattern, and silent pattern clustered together and were suggested to imply "loss of ordered growth," the cluster of loops, networks, and straight vessels to imply "fast growing clones of cells," and parallel vessels with or without cross-linking to imply "orientation of the section." Both agreed that the patterns normal, arcs, arcs with branching, parallel with cross-linking, loops, and networks carry prognostic information (6,13).
The histogenesis of vascular patterns in uveal melanoma is largely open, but they might be initiated by growing microvessels that act as a scaffold for deposition of connective tissue elements (6,11-15). Their vascular core can be verified with Ulex europaeus I lectin (5,16) or antibodies to factor VIII-related antigen (13), CD31 epitope (12), CD34 epitope (16), and other endothelial markers. The patterns can be conveniently evaluated with periodic acid-Schiff stain without counterstain, which labels their extracellular components (5,6,11,13) that have been referred to as fibrovascular (17) or periodic acid-Schiff (13) patterns.
Whether microvascular patterns have independent prognostic value has become a matter of controversy, however (6,8-10,13,17-19). In their dataset, Folberg's group (6,8-10) showed that, parallel with cross-links and network, patterns signify poor prognosis, whereas melanomas that contain only normal, silent, straight, and parallel patterns, which are characteristic of uveal nevi, have a favorable prognosis (20). In subsequent datasets, however, other prognostic factors were deemed to be more important than microvascular patterns, or the vascular patterns were claimed to have no independent prognostic value at all (13,17).
We planned a study to confirm whether the analysis of microvascular patterns in malignant uveal melanoma truly provides clinically useful information. We consider it important to solve this controversy because microvessels are logically associated with the hematogenous route of dissemination (6,9) and preliminary data suggest that microvascular patterns might be assessed clinically with noninvasive imaging techniques (21,22).
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PATIENTS AND METHODS |
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Our primary aim was to confirm or disapprove an association between microvascular patterns and survival of patients with primary malignant uveal melanoma. Our secondary aim was to determine the frequency of microvascular patterns in our patients and the level of interobserver agreement in identifying them. A third goal was to analyze the interrelationship between vascular patterns, cell type, tumor size, and tumor location in our dataset. All investigations were approved by a local institutional review board.
In their initial matched case-control study of 40 patients, Folberg et al. (5) identified the pattern of loops as the strongest prognostic indicator analyzed. In their subsequent cohort study of 234 patients, these authors reported that the pattern of networks was even more strongly associated with prognosis (6). For these reasons and also because networks consist of a series of loops making these two patterns interrelated (5), we looked for both loops and networks in the present study.
Calculation of the number of patients needed to prove a clinically significant difference between vascular patterns and survival was done by simulation (23). We based our simulation on a cohort study by Folberg et al. (6), in which 10-year cumulative probabilities of survival were 0.51 and 0.88 for patients with and without networks, respectively, with a 0.37 difference in survival. The corresponding figures for loops and no loops were 0.55 and 0.91, with an equivalent 0.36 difference in survival.
Because first reports often overestimate effects, we used in our simulation survival proportions 0.65 and 0.85 for patients with and without a vascular pattern, respectively, corresponding to a 0.20 difference in survival, which we considered to be the smallest clinically significant difference. The simulation gave a total sample size of 150 patients, divided between the two arms according to the ratio 45 : 55 that corresponds to the frequency of networks in the cohort study by Folberg et al. (6). The power of our study was 0.9 (for a two-tailed alpha of 0.05).
Study Population, Exclusion Criteria, and Histopathology of Primary Uveal Melanomas
The study population was chosen so that the minimum follow-up for patients still alive would be 15 years. This was deemed necessary to get a reliable estimate of 10-year survival, because the tails of survival curves are not stable after many patients have been censored (24) and most patients with uveal melanoma are elderly and prone to die of unrelated diseases. Diaries of the Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital were searched backward from 1981 and consecutive patients who had undergone a surgical removal of an eye because of a choroidal and ciliary body melanoma were enrolled, until the number of patients, all Caucasian, was 170 by year 1972.
During this period, removal of the cancerous eye was the standard treatment for all but the smallest uveal melanomas, and all eyes enucleated in the district of the Helsinki University Central Hospital were submitted to its Ophthalmic Pathology Laboratory, making the series essentially unselected and representative of all malignant uveal melanomas treated during that time period.
The histopathologic specimens were retrieved and new sections were cut. We adhered to the exclusion criteria of the cohort study by Folberg et al. (6) and excluded tumors rediagnosed as choroidal nevi by current diagnostic criteria (one patient), melanomas that were more than 50% necrotic (15 patients), and, by analogy, specimens in which either less than 50% of the original melanoma remained or the remaining part was entirely on the vitreal side of Bruch's membrane (16 patients). Two blocks could not be located. We did not need to exclude patients who had a second primary tumor (6) because histopathologic confirmation of the cause of death was available for all of them.
The paraffin blocks were cut at 5 µm, and the slides were randomly coded by an outside laboratory technician. The code was revealed only after both the final histopathologic and follow-up data were ready for analysis; all investigators were unaware of the outcome of individual patients until that time. The cell type was registered according to the modified Callender classification (spindle, mixed, or epithelioid) from a hematoxylin-eosin-stained section (25). If the original pathology report mentioned presence of epithelioid cells in a tumor registered as spindle cell type, this tumor was upgraded to mixed cell type. The location (choroid, ciliary body, or both), the largest basal diameter and height of the tumor, and the presence or absence of extraocular extension were taken from the original pathology reports and checked for consistency with the sections studied.
Evaluation of Microvascular Patterns
In analyzing microvascular patterns, the definitions by Folberg et al. (5,6) were adhered to. A loop was defined as a completely closed blood vessel, and the presence of one loop was sufficient to record this pattern as being present. A network was composed of at least three back-to-back loops. By definition, if networks are present, loops are present.
For the sake of consistency, all ocular specimens irrespective of the amount of melanin were bleached with 0.25% potassium permanganate followed by 5% oxalic acid (6,13,20) and stained with periodic acid-Schiff without hematoxylin counterstain to identify loops and networks (5,6).
Two observers independently examined the slides under a light microscope using a dark-green filter (5,6) (Wratten No. 58; Kodak, Rochester, NY) and recorded the presence or absence of loops and networks. Prior to analyzing the randomly coded study slides, the criteria to be used were agreed upon, and a set of slides, not included in the study series, was examined at a double-headed microscope. Disagreements in registering vascular patterns in the study series were resolved in the same manner.
In case either observer was unsure whether a pattern was truly
microvascular, he could consult adjacent sections stained for vascular
endothelium and smooth muscle, using the avidin-biotinylated
peroxidase complex method (Vectastain ABC Elite Kit; Vector
Laboratories, Inc., Burlingame, CA) as described previously in detail
(26). The primary mouse monoclonal antibodies (MAbs) QBEND/10
(lot 513; Novocastra Laboratories, Newcastle-upon-Tyne, U.K.; diluted 1
: 25) (27) to the CD34 epitope of endothelial cells and 1A4
(lot 98F4808; Sigma Chemical Co., St.Louis, MO;
1 : 8000) (28) to -smooth muscle actin
and a rabbit antiserum to factor VIII-related antigen (FVIII-Rag; Dakopatts,
Copenhagen, Denmark; 1 : 400) were commercially obtained.
To enable evaluation of immunoreaction in pigmented tumors, melanin was bleached after immunostaining with 3.0% (vol/vol) hydrogen peroxide and 1.0% (wt/vol) disodium hydrogen phosphate as described previously (29).
Follow-up Data and Histopathology of Metastases
Complete follow-up data for each patient were assembled from data retrieved from the Finnish Population and Cancer Registries, patient charts of all hospitals where they had been treated for uveal melanoma, its metastases and other malignant tumors, pathology laboratories, and death certificates. Moreover, a questionnaire concerning treatment of malignant tumors was sent to all living patients. Case histories of patients who had not been autopsied were reviewed, and the diagnosis of disseminated malignant melanoma was made if clinical findings (e.g., palpable tumor, elevated liver function tests, and abnormal liver scan) existed with a typical constellation of symptoms (e.g., abdominal pain, hepatomegaly, and rapid progression to death) without evidence of a second cancer.
Review of the clinical data revealed that one patient had cutaneous melanoma metastatic to the choroid and another had a uveal melanoma diagnosed at autopsy; they were excluded from the study, leaving 167 patients with uveal melanoma in the statistical analysis.
The original paraffin blocks from 31 of 33 surgical biopsy specimens and from 27 of 29 autopsies could be retrieved from other laboratories. Eleven fine-needle aspiration biopsy specimens had been taken, but most of these slides could not be retrieved. Altogether, we could verify 58 tumor deaths by histopathology.
Immunoperoxidase staining was used to confirm metastatic melanoma and secondary cancers. The primary mouse MAbs HMB-45 (lot 0024b; DAKO A/S, Copenhagen, Denmark; diluted 1 : 200) to immature melanosomes that labels more than 90% of primary and metastatic uveal melanomas (30-32), MAb CY-90 (lot 49F-4815; Sigma Chemical Co.; 1 : 3000) to cytokeratin 18 that is expressed by adenocarcinomas, but can also be found in a minority of cells in uveal melanomas (26), MAb Vim 3B4 (lot 114544324-01; Boehringer-Mannheim GmbH, Mannheim, Germany; diluted 1 : 50) to vimentin that labels uveal melanomas (26) but may also label cells in some carcinomas, and MAb E29 (lot 078; DAKO; diluted 1 : 25) to epithelial membrane antigen produced by adenocarcinomas but not by melanomas (33) were commercially obtained. A tumor was accepted as a melanoma if it reacted for HMB-45, vimentin, or both, but not for cytokeratin 18 and epithelial membrane antigen, and as a carcinoma when the antigenic profile was the opposite.
Of the 58 reanalyzed metastatic tumors, the original diagnosis was found to be incorrect in five (9%; 95% confidence interval [CI] = 3-19). Three amelanotic metastases from uveal melanoma had been misdiagnosed as metastatic carcinoma. Conversely, a hepatic metastasis from a carcinoma had been misdiagnosed as metastatic melanoma. Finally, one biopsy specimen had been correctly read as metastatic mucocellular carcinoma, but specimens taken at autopsy had been erroneously read as metastatic uveal melanoma.
Statistical Analysis
All statistical analyses were carried out with the BMDP PC-90 Statistical Software program (BMDP Statistical Software, Cork, Ireland). Descriptive statistics for normally distributed variables are given as mean and standard deviation and for other variables as median and range.
Two-tailed Fisher's exact test and Pearson's chi-squared test with Yates' continuity correction were used to compare proportions in 2 x 2 and larger contingency tables, respectively, medians were compared with the Mann-Whitney U test, and kappa statistic was used to estimate chance-corrected interobserver agreement (34).
Univariate analysis of melanoma-specific survival time data was based
on the Kaplan-Meier product-limit method, and survival curves were
compared with the Mantel-Cox test (24). In this analysis,
patients judged to die of other causes were censored at their time of
death. To guard for the possibility that these persons were more or
less likely to have progression of melanoma than other patients,
all-cause mortality was also analyzed. Moreover, equality of follow-up
between groups was ascertained by comparing Kaplan-Meier curves with
reverse censoring (24). The effect of loops and networks was
analyzed both separately and by a combined variable that considered
networks to be an advanced stage of loops with three categories: no
loops, loops without networks, and networks. Multiple pairwise
comparisons were done, adjusted by the Bonferroni correction
(34). The cell type was collapsed into two categories based on
the presence of epithelioid cells (spindle versus nonspindle)
(6,13), and tumor location was dichotomized according to the
presence of ciliary body involvement (6,9,17). The largest
basal diameter was divided in three categories: small (10 mm),
medium (>10-15 mm), and large (>15 mm) (6).
Multivariate analysis of survival time data was based on the Cox proportional hazards model (24,35). The effect of loops and networks was analyzed by the combined categorical variable that considered networks to be an advanced stage of loops. The largest basal diameter was analyzed as a continuous variable, similar to tumor height and age of the patient that were also included in multivariate modeling. Forward stepwise regression was used to identify independent prognostic factors (24,35). The regression coefficients and hazard ratios (HRs) with their 95% CIs were calculated (24). The assumption of proportional hazards was ascertained with complementary log plots (24).
Possible interaction between vascular patterns and other variables that entered the stepwise model were tested by comparing models that included the combined categorical variable that considered networks to be an advanced stage of loops, a confounding variable, and a product term involving these two variables (35). Regression coefficients and HRs were calculated after stratification by those confounding variables that had interaction with the combined vascular pattern variable.
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RESULTS |
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Of the 167 consecutive uveal melanomas, 134 (80%) fulfilled the
inclusion criteria for analysis of vascular patterns. The clinical
characteristics of the included patients did not differ from those of
the excluded patients (Table 1).
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Microvascular loops were more common in tumors that involved the
ciliary body (P = .040, Fisher's exact test; Table
2) and in tumors that contained epithelioid cells
(P = .0031), and they had a tendency to become more frequent
when the largest basal diameter increased (P = .069,
chi-squared test for trend). Networks were more common in tumors with
epithelioid cells (P = .024, Fisher's exact test; Table 2
).
Networks-to-loops ratio did not increase consistently with the largest
basal diameter, being 0.53 for small, 0.63 for medium, and 0.58 for
large melanomas.
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Univariate Analysis of Melanoma-Specific Survival
At the end of the follow-up, 44 (26%) of 167 patients were alive
without evidence of recurrent melanoma, 76 (46%) had died of
metastatic uveal melanoma, nine (5%) had died of other cancers, and
37 (22%) had died of other reasons (Table 1).
The 10-year cumulative melanoma-specific probability of survival of the
134 patients included in the analysis of vascular patterns did not
differ from that of the 33 patients excluded from the analysis due to
technical reasons (0.60 versus 0.54, P = .39; Mantel-Cox
test; Fig. 2, A). The survival of patients with
tumors with epithelioid cells (nonspindle mixed and epithelioid cell
tumors as compared with spindle cell tumors; 0.35 versus 0.74,
P<.0001; Fig. 2
, B), tumors involving the ciliary body
(0.35 versus 0.68, P = .0003; Fig. 2
, C), and large tumors
(P = .0001; Fig. 2
, D) was worse than that of patients
with tumors that lacked these characteristics. The results were comparable
when all-cause mortality was compared (0.25 versus 0.57, P =
.002 for epithelioid cells; 0.29 versus 0.51, P = .014 for
ciliary body involvement; and P = .0001 for tumor size).
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Again, the results were similar when all-cause mortality was compared
(0.34 versus 0.62, P = .001 for loops; 0.30 versus 0.54,
P = .009 for networks; and 0.30 versus 0.39 versus 0.62,
P = .0035 for the combined variable; Fig. 2, H).
Reversed
censoring confirmed that patients were censored from all univariate
analyses independent of their assignment to the groups being compared,
except for the largest basal diameter (P = .017).
Multivariate Analysis of Melanoma-Specific Survival
In univariate Cox regression, presence of loops and presence of
epithelioid cells had the strongest relationship with melanoma-specific
survival (2 = 22.9 and 20.7, respectively; Table
3).
Considering networks to be an advanced stage of
loops gave similar results as compared with loops analyzed separately
(
2 = 22.8 versus 22.9; Table 3
). Networks,
largest basal
diameter, ciliary body involvement, tumor height, and age analyzed as
univariate factors also had a statistically significant relationship
with prognosis (Table 3
).
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In interaction analysis, product terms involving the combined vascular patterns variable and the confounding variables epithelioid cells (likelihood ratio = 493.4 - 489.4, P = .047 chi-squared, 1 df) and ciliary body involvement (likelihood ratio = 496.8 - 492.3, P = .0347) were statistically significant, whereas product term involving largest basal diameter was not quite statistically significant (likelihood ratio = 495.4 - 492.0, P = .0664).
When Cox regression was restricted to melanomas without epithelioid
cells, combined vascular patterns (P = .0001; HR = 2.45) were
associated with metastatic death, whereas the relative statistical
significance of largest basal diameter and ciliary body involvement
diminished (Table 4). In contrast, when melanomas with epithelioid
cells were analyzed, largest basal diameter became the predominant
predictor of melanoma-specific survival (P = .0071; HR = 1.16,
for each 1-mm increase) (Table 4
).
When Cox regression was restricted to choroidal melanomas, combined
vascular patterns (P = .0002; HR = 2.17) and cell type
(P = .032; HR = 2.09) were primarily associated with death
from metastases, whereas the presence of epithelioid cells (P
= .0037; HR = 4.63) became the predominant predictor of
melanoma-specific survival in a model for melanomas with ciliary body
involvement (Table 4).
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DISCUSSION |
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Networks were found in 35% (95% CI = 27-44) of melanomas studied, a figure that is somewhat smaller than that reported by Folberg et al. (6) (45%; 95% CI = 39-52). The frequency of networks was even smaller in the series by Foss et al. (13) (25%; 95% CI = 17-34) and Seregard et al. (19) (19%; 95% CI = 12-27). In the dataset of Rummelt et al. (9), networks were more frequent in melanomas that involved the ciliary body as compared with choroidal melanomas (67% versus 39%), a difference we did not observe (36% versus 33%). Because tissue processing and microscopy were done identically in all four studies, other factors must be invoked to explain the difference.
The five series mentioned had dissimilar inclusion criteria. The series by McLean et al. (17) and Foss et al. (13) were enriched on purpose with patients who died of melanoma (36), while the study by Seregard et al. (19) had a case-control design. Because loops and networks signified poor prognosis in these studies, it is unlikely that lower frequencies of loops and networks were due to random chance. The inclusion rate of these studies ranged from 11% to 55% (6,13,17,36), whereas we were able to analyze 80% of consecutive patients.
Another explanation for the variation in frequencies of loops and networks may be the criteria used in identifying them. We counted loops of all sizes, provided that they had a microvascular core. In the dataset of Folberg et al. (5), the diameter of loops varied 10-fold, suggesting that similar criteria were applied. From the correspondence by Folberg et al. (6,12,37) and Foss et al. (13), it is evident that they gave different weight to associated fibrous tissue. Our study confirms that it is crucial to recognize microvascular patterns that lack a substantial fibrous component (12,37), but it also showed that infiltrated ciliary muscle can imitate microvascular patterns. Our most frequent source of disagreement was, however, whether a loop was completely closed or not (17).
These potential sources of disagreement notwithstanding, the interobserver agreement in our study was good (34). We agreed on presence of loops and networks in 85% of cases, with kappa values 0.70 and 0.67, respectively, which were similar to the estimates by Folberg et al. (kappa values, 0.67 and 0.59, respectively) (6). Microvascular loops and networks can thus be identified in outside laboratories with a reliability comparable to that of the center that developed the method.
In our univariate analysis, the cumulative probability of survival was worse when loops and networks were found as compared with tumors without these vascular patterns. The difference in 10-year survival was 0.38 for loops and 0.31 for networks, comparable to the 0.36 and 0.37 differences that Folberg et al. (6) observed, respectively (6). In the study by McLean et al. (17), the difference for loops was 0.33. Loops and networks were roughly as good in predicting death from melanoma as were the site, size, and cell type of uveal melanoma, and vascular patterns were the strongest univariate prognostic factor, again corroborating the results of Folberg et al. (6) and Mehaffey et al. (8).
In contrast to the series by Folberg et al. (6) and Seregard
et al. (19), loops instead of networks were the strongest
overall indicator of tumor death (Table 5). In fact,
during the first 5 years from treatment, no difference was observed in
survival between tumors with loops only as compared with those with
networks. It is important to note that no series has shown a difference
in the overall ability of loops and networks to differentiate patients
with good and poor prognosis by univariate analysis (6,19).
Random chance related to different inclusion criteria, rates, and case
mix may have determined whether loops or networks entered the
multivariate model in these series. A combined variable that considers
networks to be an advanced stage of loops as used in the present
analysis avoids this potential problem.
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We have confirmed that analyzing microvascular patterns helps the pathologist to differentiate uveal melanomas into those with favorable and poor prognosis (5,6). In particular, they might help to predict which choroidal and spindle cell tumors that generally have a more favorable prognosis eventually metastasize. Our results support the usefulness of recording microvascular patterns in pathology reports (6,17) and encourage the development of noninvasive methods, such as ultrasound power spectrum analysis (21), color Doppler imaging (16), and indocyanine green angiography (22), which might enable visualization of these patterns before treatment. Finally, microvascular patterns might be worthwhile to study in other cancers that spread predominantly hematogenously.
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NOTES |
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