- Email: [email protected]
The Prognostic Value of Tumor Blood Vessel Morphology in Primary Uveal Melanoma
The Prognostic Value of Tumor Blood Vessel Morphology in Primary Uveal Melanoma Robert Folberg, MD,1,2 Volker Rummelt, MD,l Rita Parys-Van Ginderdeuren, MD/ Taekyu Hwang, MS,3 Robert F. Woolson, PhD,3 Jacob Pe'er, MD,4 Lynn M. Gruman, HT(ASCP)l
Background: It is possible to identify at least nine vascular patterns in melanomas of the ciliary body and choroid from histologic sections. An association between the presence of at least one closed vascular loop and death from metastases was shown in a matched-pair, case-control study of 40 patients whose eyes were removed for ciliary body or choroidal melanomas. Methods: Two independent observers who were masked to the follow-up of patients examined histologic preparations of 234 eyes removed for ciliary body or choroidal melanomas for the presence of each of the tumor vascular patterns. Statistical analyses included tests for interobserver reliability, Kaplan-Meier survival curves, and the fitting of Cox regression models. Results: The detection of each of the nine vascular patterns is highly reproducible. The Cox model indicates that the presence of vascular networks, defined as at least three back-to-back closed vascular loops, is the feature most strongly associated with death from metastatic melanoma. Other significant factors in the Cox model include (in descending order of importance) largest tumor dimension, mitoses, the parallel with cross-linking vascular pattern, age, the presence of tumor-infiltrating lymphocytes, and male gender. Conclusions: The presence of vascular networks provides the most significant association with death from metastatic melanoma of all variables tested. The presence of this pattern should be recorded on pathology reports. If it becomes possible to detect this vascular pattern clinically using a noninvasive imaging technique, then ophthalmologists may be able to determine the likely biologic behavior of a melanoma before resorting to the removal of tissue. Ophthalmology 1993;100:1389-1398
The treatment of most cancers is based on a histologic assessment of the tumor's biologic behavior (grade) and the extent of spread from the primary site (stage).
Ophthalmologists must infer the biologic tumor grade from clinical observations: the largest tumor dimension (LTD) (especially the largest diameter in contact with the
Originally received: January 12, 1993. Revision accepted: March 8, 1993.
Dr. Parys Van-Ginderdeuren is currently at the Universitaire Ziekenhuizen St.-Rafael, Leuven, Belgium. Supported by National Institutes of Health grant EY07043, Bethesda, Maryland, and in part from an unrestricted grant from Research to Prevent Blindness, Inc, New York, New York. Dr. Rummelt is the recipient of grant Ru464/3-1 from the Deutsche Forschungsgemeinschaft, Germany. Reprint requests to Robert Folberg, MD, University of Iowa, 100 Medical Research Center, Rm 233, Iowa City, IA 52242-1182.
I
Department of Ophthalmology, University of Iowa, Iowa City.
2
Department of Pathology, University of Iowa, Iowa City.
Division of Biostatistics of the Department of Preventive Medicine, University of Iowa, Iowa City. 3
Department of Ophthalmology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
4
1389
Ophthalmology
Volume 100, Number 9, September 1993
Figure 1. The nine morphologic vascular patterns of primary ciliary body and choroidal melanomas (all photomicrographs: periodic acid-Schiff without hematoxylin; original magnification, X280). Top left, normal: tumor cells grow around normal choroidal vessels that are either filled with blood (n) or vacant (N). Top right, silent: no normal vessels or new vessels are identified. Center left, straight pattern: a straight vessel connects with a normal vessel (N). Center right, parallel: straight vessels are oriented in a parallel array. Bottom left, parallel with cross-linking: the cross-links are identified by arrowheads. Bottom right, arcs: arcs are fragments of curved vessels or incompletely closed loops. (Fig 1 continues)
sclera), I the location of the tumor (tumors situated anteriorly are more associated with subsequent metastases than those located posteriorlyj.i" and the presence or absence of extraocular extension' as detected by ultraso-
1390
nography. After an eye has been removed, pathologists may assign a true histologic grade (i.e., the likelihood of a patient dying of metastatic melanoma) by considering the following features: cell type,6,7 cytomorphometric as-
Folberg et al
Uveal Melanoma Tumor Vessels
Figure 1, continued. Top, arcs with branching: dichotomous branching from fragments of incompletely closed loops. Center, loops: an island of tumor is surrounded by a large, closed vascular loop (L) which is to the right of a vacant, normal vessel (N). Bottom, networks: back-to-back adjacent loops. Notice the relationship of the network to a normal vessel (N).
sessments of the nucleoli.r '? and nucleolar measurements combined with the largest area of scleral contact!' (along with those features that are also detectable clinically: tu-
mor size, location, and presence of extraocular extension). Although fine-needle aspiration biopsies of uveal masses may distinguish between primary melanomas and tumors metastatic to the uvea, these specimens do not accurately reflect the cell type identified from histologic sections." Also, there is no association between cytomorphometric analyses of nucleolar dimensions measured from fineneedle aspiration biopsy specimens and sections from eyes that have been removed. 13,14 Therefore, the management of uveal melanomas would be enhanced if a noninvasive method were available to grade the likelihood of a tumor generating metastases. Recently, the dermatologic community developed noninvasive techniques for grading cutaneous melanoma, Using ultrasonographic techniques, it is possible to detect tumor vascularity in cutaneous melanomas15 that correlates well with corresponding histologic sections.l'"!" In addition, there is an association between the density of tumor vessels and prognosis in some cutaneous melanomas. 15,18-20 Similar relationships between blood vessel counts and prognosis have been established for adenocarcinomas of the breast,21-23 non-small cell carcinoma of the lung," and carcinomas of the prostate." The ophthalmic community also has been interested in the ultrasonic detection oftumor vascularity. Coleman and co-workers" correlated acoustic patterns with the Callender cell type, but they did not discover an association between vascularity and cell type." Miller and coworkers'" reported an association between the vascularity of uveal melanomas on tissue sections and prognosis, but this relationship has not been confirmed (K. Egan, personal communication; May 1992). Carnochan and associates 19 discovered that the density of tumor vessels did not correlate well with survival for cutaneous melanomas measuring between 0.85 and 1.25 mm. These investigators suggested that "the assessment of other features of the tumour vasculature may provide a useful complement to simple morphometry." Instead of measuring tumor vessel density, Folberg and coworkers" identified nine vascular patterns in tissue sections of uveal melanomas; in a matched-pair case-control study of 40 eyes, the vascular pattern most closely associated with death from metastatic melanoma after enucleation was the presence of one or more closed vascular loops. The presence of these loops also was associated with mitotic figures and epithelioid cells, histologic features associated with an unfavorable outcome after enucleation. We now report the relationships between tumor vascular patterns and survival in a larger series of eyes removed for uveal melanoma.
Materials and Methods We asked the Tumor Registry of the University of Iowa to identify all patients who had a diagnosis of "intraocular melanoma." Of the 7 80 cases identified, 199 did not have any pathologic material on file in our laboratory. Because we needed to use special stains to demonstrate the microcirculation of these melanomas, we required the use
1391
Ophthalmology
Volume 100, Number 9, September 1993
of paraffin block to recut and stain slides according to the methods described below. We could not locate the blocks of 200 cases. An additional 147 cases were eliminated for one or more ofthe following reasons: the blocks that were available did not contain complete sections of tumor that were representative in size compared with the dimensions recorded in the gross pathology section of the pathology report, pure iris melanomas, eyes that had received any form of pre-enucleation treatment such as radiation, eyes from patients identified as having more than one cancer (including bilateral uveal melanoma), eyes from patients enrolled in the Collaborative Ocular Melanoma Study." nevi, and tumors more than 50% necrotic. Thus, 234 tumors met the criteria for entry into this study. The Iowa Tumor Registry provided follow-up data for all cases. At least two adjacent paraffin sections were cut at 5 to 6 Jlm. One section was stained with hematoxylin-eosin and was treated by permanganate bleaching if nuclear features were obscured by melanin. One slide was stained with periodic acid-Schiff (PAS) without hematoxylin counterstaining (with permanganate bleaching in cases of heavily pigmented tumors) and was viewed with a green filter; the accuracy of this technique compares favorably with more specific methods of identifying vessels in the uveal melanoma microcirculation." One of us (RF) recorded the following information from the hematoxylin-eosin slide: the tumor location, the largest tumor dimension (there is no significant difference between using tumor dimension measurements from glass slides and gross pathology reports in studies of patient survival:"), the number of mitotic figures per 40 highpower fields (X40 objective using a X10 eyepiece), the cell type using the modified Callender classification," the presence of 100 lymphocytes per 20 high-power fields." and the presence of extraocular extension or a diffuse growth pattern. Two of us (VR and RPV) who were not involved in the initial description of the nine morphologic patterns of tumor vessels in uveal melanoma" independently reviewed the slides stained with PAS without hematoxylin and recorded the presence or absence of each of the following nine vascular patterns" (Fig 1): 1. Normal Pattern: Tumor cells grow around normal
2. 3. 4. 5.
1392
choroidal vessels without compressing them. Three normal vessels must be detected beneath Bruch's membrane at Xl 0 magnification with a X10 ocular for this pattern to be recorded as present (dilated normal vessels seen above Bruch's membrane have been attributed to stagnant venous return"), Silence: No normal or new tumor vessels are present in a Xl 0 field viewed with a X10 ocular. Straight Vessels: Randomly distributed straight vessels lack dichotomous branching and are not cross-linked to each other. Parallel Pattern: Straight vessels, lacking dichotomous branching or cross-linking, are aligned parallel to each other. Parallelwith Cross-linking: Parallel vessels are crosslinked to each other; this pattern may resemble switching tracks in a rail yard.
6. Arcs: Arcs are incomplete loops. We did not obtain serial sections through the tumor to discover if arcs became loops in deeper sections. 7. Arcs with Branching: Arcs show dichotomous branching. 8. Loops: A loop is completely closed. The presence of one loop is sufficient to record this pattern as present. 9. Networks: A network is composed of least three back-to-back closed loops. By definition, if networks are present, loops are present. None of the investigators examining the slides knew the follow-up status. The investigators recorded the presence or absence of each ofthe nine vascular patterns from the slides stained by the PAS without hematoxylin method independently; the observations for each pattern in each case were compared for interobserver reliability. Discrepancies between the two observers were resolved by consensus, and this consensus observation was used in further statistical analyses. We tested the interobserver reliability for the identification of each vascular pattern using the chance-corrected measure of agreement (Kappa statistic). 34 The continuityadjusted chi-square test was used when appropriate to explore possible associations between individual vascular patterns and the following features derived from the histologic examination: tumor location, cell type, mitoses, the presence of more than 100 lymphocytes per 20 highpower fields, extraocular extension, and a diffuse growth pattern. We tested for an association between each vascular pattern and location with three types ofcomparisons: (1) all tumors confined to the choroid versus tumors having a component in the ciliary body, (2) tumors in the choroid posteriorly versus tumors in the choroid anteriorly versus tumors involving the ciliary body, and (3) tumors of the choroid posteriorly with tumors of the choroid anteriorly. We tested the possible association between each vascular pattern and cell type in two ways: (1) by using three categories of the modified Callender classification7 (spindle cell melanoma, mixed cell type and epithelioid), and (2) the presence or absence of any epithelioid cells (spindle cell melanoma versus a combination of mixed cell type and epithelioid tumors). 35 The primary outcome variables for individuals whose eyes are included in this study consist of (I) the time to death from metastatic melanoma or from other causes, or (2) the time to follow-up for those patients who are still alive. The analysis described here focuses on the time to death from metastatic melanoma. We treated time-todeath from other causes, time-to-follow-up for living patients, and time to last contact for patients reported as "lost to follow-up" as censored times in the data analyses. The primary statistical model fit is the Cox proportional hazards model. This model is appropriate for the analysis of failure time or mortality data and is sufficiently flexible to handle multiple covariables. The primary assumption involved in this model is that of proportional hazards, namely the hazard function for an individual with one set of characteristics is proportional to the hazard function
Folberg et al . Uveal Melanoma Tumor Vessels Table 1. Agreement between Two Independent Observers for Each Vascular Pattern
Pattern
% Agreement
Normal Silence Straight Parallel Parallel with cross-linking Arcs Arcs with branching Loops Networks
94 91 92 83
77 81 83
84 80
Chance Corrected Measure of Agreement (kappa statistic]"
-0.028 0.807 0.056 0.479 0.530
0.444 0.611 0.667 0.591
• p < 0.0001 for all observations except the normal pattern. In this pattern, nearly every observation is in one category so there is little variability; the % agreement may be a more meaningful statistic for this variable.
for an individual with another set of characteristics. This ratio is commonly thought of as the relative risk. Stepwise regression techniques were used to fit several models involving the selection of variables; score chisquare statistics" with significance levels of 0.05 were used for entry and removal in the stepwise model building. We fit survival curves for individual groups using the KaplanMeier method." We calculated the log-likelihood ratio statistic (-210gL) to test several regression coefficients simultaneously and to compare competing models. Wald chi-square" was used to assess final model coefficients. In all instances, the analyses involved fitting Cox regression models." We used the SAS PH REG procedure."
Results The tumors came from 106 men (45.3%) and 128 women (54.7%).The agesof patients at the time of diagnosis ranged Table 2. Frequency Distributions for Vascular Patterns'" in Choroidal and Ciliary Body Melanomas Cases Identified (n = 234) Vascular Pattern
No. (0/0)
Normal Silent Straight Parallel Parallel with cross-links Arcs Arcs with branching Loops Networks
226 (97) 104(44)
226 (97) 198(85) 140(60) 182 (78) 159 (68) 131 (56) 106(45)
from 18 to 88 years (median, 59 years). Follow-up ranged from 6 months to 39.7 years (median survival, 7.6 years). Follow-up information showed that 101 patients (43.2%) were alive without any evidence of cancer, 51 (21.8%) had died without evidence of metastasis, 67 (28.6%) had died of metastatic melanoma, and 15 (6.4%)were lost to followup. The LTD in contact with the sclera" ranged from 5 to 23 mm (median, 12 mm). Sixty-seven tumors (28.6%) were small « 10 mm in diameter), 117 tumors (50%) were medium-sized (> 10 mm but < 15 mm in diameter), and 50 tumors (21.4%) were large (~15 mm in diameterj." One hundred thirty-two tumors (56.4%).were located in the choroid posterior to the equator, 48 (20.5%) were located in the choroid anterior to the equator without involvement of the ciliary body, 47 (20.1%) involved the choroid and ciliary body, 2 (0.9%)involved only the ciliary body, and 5 (2.1%) involved the choroid, ciliary body, and iris. One hundred twelve tumors (47.9%) were classified as spindle cell melanomas, 103 (44%)were classifiedas mixed cell type, and 19 were classified as epithelioid melanomas (8.1%).7 At least one mitotic figure was identified in 112 tumors (47.9%). At least 100 lymphocytes per 20 highpower fields'? were identified in 67 tumors (28.6%). Extraocular extension was identified in nine tumors (3.8%), and seven (3%) were classified as diffuse. The identification of each vascular pattern was highly reproducible (Table 1). Table 2 summarizes the frequency distribution for each vascular pattern. Parallel with crosslinking, arcs, arcs with branching, loops, and networks are more likely to be detected in the ciliary body than in the choroid; parallel with cross-linking, arcs, arcs with branching, loops, and networks are more likely to be detected in the choroid anterior to the equator than in the choroid posteriorly. Chi-square tests showed that no vascular pattern had a significant association with extraocular extension or a diffuse growth pattern. A statistically significant association was identified between the presence of epithelioid cells" (spindle cell melanomas versus mixed and epithe-
1393
Ophthalmology
Volume 100, Number 9, September 1993
lioid cell melanomas) and the following vascular patterns: parallel (P = 0.001), and arcs, parallel with cross-linking, arcs with branching, loops, and networks (all P < 0.000 I). The absence of epithelioid cells was associated with the silence pattern (P = 0.007). The presence of at least 100 tumor-infiltrating lymphocytes per 20 high-power fields was associated with the following vascular patterns: parallel with cross-linking (P = 0.001), loops (P = 0.004), and networks (P = 0.00 I). Figure 2 shows Kaplan-Meier survival curves generated from deaths due to metastatic melanoma according to location, LTD, cell type, parallel with cross-linking, loops, and networks. Briefly, at the lO-year follow-up visit, the survival of patients whose tumors lack the vascular patterns of parallel with cross-linking, loops, and networks is 91.7%, 91.1 %, and 88.3%, respectively, versus 56.9%, 55.4%, and 50.7% if these patterns are present. KaplanMeier survival curves for all patient deaths are similar to those illustrated for deaths from metastatic melanoma. When considering all variables, including the vascular patterns, the complete model (i.e., the one that includes all variates) suggested that statistical collinearity exists between two of the vascular patterns. These patterns (arcs and arcs with branching) were dropped from the analysis. We initiated the stepwise regression after these variables were deleted. Table 3 summarizes the results from fitting the stepwise regression procedure and includes a summary of the stepwise modeling at each step and the individual chi-square statistic from the score procedure. The most important variable (and the one to enter the model first) is the network pattern (chi-square = 40.84; P = 0.0001). At the second step, the next most important variable, given that the network pattern was in the model, was LTD (chisquare = 14.64; P = 0.0001). Table 3 is self-explanatory thereafter. None of the variables entered in the preceding steps was removed (i.e., each retained its importance in the model). After the seventh step, no additional variables reached significance for entry into the model. Therefore, the final model included networks, LTD, mitoses, parallel with cross-linking, age, tumorinfiltrating lymphocytes, and the patient's sex (male gender). Table 4 summarizes the estimated Cox model regression coefficients. Note that each estimate is a positive number, indicating that the presence of networks, at least one mitotic figure and the parallel with cross-linking patterns are associated with a higher risk of death from metastatic melanoma. Table 4 also includes standard errors along with chi-square statistics. Each of these partial chisquare statistics has a very small P value, indicating the importance of these terms in the model. Table 4 also includes the value of the log likelihood with its seven degrees of freedom. This stepwise modeling suggested that these seven variables were the most important; of these seven, the two most important variables were the presence of vascular networks and the LTD.
Discussion In a matched-pair case-control study of 40 eyes removed for ciliary body or choroidal melanoma, we showed that
1394
the presence of at least one closed vascular loop was strongly associated with death from metastatic melanoma." Our current study confirms that vascular patterns are more strongly associated with death from melanoma after enucleation than the LTD, location, cell type, mitotic count, and tumor-infiltrating lymphocytes. In the Cox model considering deaths from melanomas, the presence of networks is the factor most strongly associated with death from metastatic melanoma. In the Cox model considering all deaths, the presence of networks is also the most significant variable. Parallel with cross-linking vessels may be a variant of networks: if vessels cross-link at more than one place, they form elongated loops. Ifthese stretched-out loops are arranged back to back, then they form elongated networks. It is not surprising that this multivariate analysis offactors shows networks and parallel with cross-linking to be more significant than loops, the factor identified as most significant in our univariate analysis": networks are composed of loops. By examining tissue sections from glass slides, we have only sampled a two-dimensional plane. There is a heterogeneous distribution of these patterns in two dimensions (i.e., when viewed on the surface of a glass slide), so it is reasonable to expect a heterogeneous distribution of these patterns deeper in the tissue block." Not only may some patterns be present or absent in deeper planes, it is possible that arcs and arcs with branching become loops when imaged in three dimensions: the detection of both arcs and arcs with branching in two-dimensional tissue sections is associated with death from metastatic melanomas in univariate analyses but is not significant in multivariate analysis. It is important to realize that tumor vessels develop in three dimensions. In some studies, investigators merely counted the number of blood vessel cross-sections per unit area (vessel density) and linked these counts to outcome (i.e., breast cancer,21-23 some forms of cutaneous melanoma,15,18,19,28 and prostatic cancer"), It is quite possible that the same vessel is counted more than once because vessels may weave in and out of section planes. In addition, the technique of counting tumor vessel cross-sections may not even be applicable to uveal melanoma: how often would a vessel be counted if it were part of a vascular network of closed loops? It is noteworthy that cell type and tumor location, variables described previously as important associations with death from metastatic melanoma.v" did not appear in the stepwise modeling and in any step along the way. Networks have an effect beyond any that may exist due to cell type when both variables are considered in any statistical formulation. From any point of view for these data, the presence of networks is the more dominant characteristic and the one that accounts for the most statistical variation. Two of the variables that appear in our model, age and sex, are not often cited as risk factors for death from metastatic uveal melanoma. However, Jensen'" also observed that patients older than 40 years of age at the time of treatment had a worse prognosis than younger patients.
Folberg et al . Uveal Melanoma Tumor Vessels Z
9
t! Z
::;J
:.r..
1.0
',-
",
' \'l.\,
"L
0.8
::;J
._._ _ .~.M_. __.
-L - L -
0.6
~
f::2
=
p-vatu e
P::l
0.8
::;J
0.6
'\
\
·L __.
ell
..... ._.... _,
i.
0.4
p-value
2: > ~
0.2
0.2
:J
en 0.0
0.0 10
20
15
1.0
u
30
25
35
a
40
. _ --
Spindle
....... ...._.
Mixed
-
Epithelioid
-
-
0.8
1.0
:J
0.8
No Pa rallel Cross
---~
't..
'---1--,
p-value
p-vatua
0.4
.0002
i5
....l
>
0.4
:>
0.2
0.2
~
::;J
:J
en
en 0.0 0
10
15
20
30
J 25
35
0.0 0
40
10
15
TIME (YEARS)
1.0
~_ _......._
u
~
_
-
.
-
L._.•._m._ _
1
No LOOP --
__.__ _ _
With loop
I
Z
6z
_ __••...__..__.. ..
0.8
~
::;J
p-value
0.6
§
i5
40
~
~--- l
0.8
---
·L
..
.
NoNo~
With NetwOrk
..._.._.._..__...
I ._
0.6
p-value
.000 1
0.4
....l
~
0.2
0.2
~
:J
::;J
en
35
~
~ 0
0.4
....l
::= > ~
1-
1.0
E9
~
E-
30
Z
.0001
E9
en
25
20
TIME (YEARS)
Z
0 1==
.0001
P::l
~ en
Z
_ ._---- - -- _.... _----- _.
0.6
:J
....l
Z 0 1==
Wi th Parallel Cross
----I
1==
0.6
::=
> ~
40
35
~
~
s
30
25
Z 0
E-
en
Z 0
C Z
Z
E9
20
15
10
TIME (YEARS)
0
§
.000 1
TIME (YEARS)
it
._._.._
_.•
....l
0
z
.._..__....
._._....-.... ,,
i5
::;J
Z 0 1==
L.T.D. ( 10-15) L.T D. ( > 15 )
, _ _ .__ ,
E0.4
en
it E9 ~
.0004
Cl ....l
::= > ~
1.
Z 0 1==
_
L.T.D . ( <= 10 )
1.0
C z
Ciliary Body -'~-'"\.t...-..,
Z
0 1==
Z 0
Choroid
ell
0.0 0
10
15
20
25
TIME (YEARS )
30
35
40
0.0 0
10
15
20
25
30
35
40
TIME (YEARS)
Figure 2. Kaplan -Meier sur vival curves for deaths from metastatic melanoma. Top left, location : tumors involving th e ciliary bod y versus tum or s confined to the choroid. Top right, largest tumor dimen sion (L.T .D.): small melanomas « 10 mm) versus medium-sized melan om as (10- 15 mm ) versus large melan omas (> 15 mm). Center left, cell type: spindle cell melanomas versus melan om as of the mixed cell type versus epithelioid melanomas. Center right, melanomas without the parallel with cross-linking pattern versus tumors with this patt ern. Bott om left, melanomas with out loops versus melanomas with loops . Bottom right, melanomas without vascular network s versus melan omas with netw orks.
1395
Ophthalmology
Volume 100, Number 9, September 1993 Table 4. Regression Coefficients and Standard Errors*
Table 3. Summary of Stepwise Regression Procedures Variable
Step
Networks Largest tumor dimension Mitoses Parallel with cross-linking Age > 100 tumor-infiltrating lymphocytes/20 HPF Sex
1 2 3 4 5 6 7 HPF
=
P
Variable
Estimate
SE
Wald Chi-square
P
14.64 9.06 10.32 6.07 5.30
0.0001 0.0001 0.0026 0.0013 0.0137 0.0214
1.056 0.108 0.148 1.172 0.025 0.642
0.3076 0.0387 0.0375 0.4148 0.0095 0.2683
11.78 7.75 15.50 7.98 7.13 5.73
0.0006 0.0054 0.0001 0.0047 0.0076 0.0167
4.57
0.0172
Networks Largest tumor dimension Mitoses Parallel with cross-linking Age > 100 tumor-infiltrating lymphocytes/20 HPF Male gender
0.536
0.2533
4.48
0.0343
Score Chisquare
40.84
high power field.
Jensen'? also noted that the survival for women with uveal melanoma was more favorable than for men. It will be necessary in a future study to evaluate the effect of vascular patterns on survival when cytomorphometric measurements'"!" are entered into a multivariate model. However, even if cytomorphometric measurements prove to have a greater effect on mortality than the presence of networks, these measurements are only useful after the eye has been removed.P:'" On the other hand, if it is possible to develop a noninvasive technique to detect vascular networks or parallel with cross-linking patterns (by ultrasonic acoustic microscopy.P-":" Doppler ultrasonography.f or other imaging techniques':'), then it may be possible to assign a histologic tumor grade in vivo. We recommend that the presence or absence of these patterns be recorded on histopathology reports. Our current study suggests that the classification system of vascular pattern can be taught easily to others: the two investigators (VR and RPV) in this study who graded each tumor independently for the presence or absence of each vascular pattern did not participate in our pilot study." Each of these individuals mastered the identification of vascular patterns within I day. There is an extremely high degree of agreement between both of the observers, suggesting that the use of the vascular patterns as an indicator of the prognosis after enucleation is highly reproducible. In addition, the laboratory techniques for the detection of these vessels may be applied easily in any pathology laboratory. We previously showed a correspondence between the modified PAS (without hematoxylin) stain used in this study and the specific method of detecting tumor blood vesselsin uveal melanomas (using the confocal laser microscopy and fluorescent Ulex europaeus I., a lectin specific for the identification of vascular endotheliumr"; the uvea lacks lymphatics. A prominent basement membrane zone frequently surrounds tumor blood vessels,29,45-47 a histologic feature that accounts for positive staining of these vessels with PAS. There are some drawbacks to the simple technique of applying PAS without hematoxylin staining to uveal melanomas to detect tumor blood vessels. First, staining sec-
1396
.
= standarderror; HPF = high power field. -2logL = 674.079; df = 7; modelchi-square = 86.850.
SE
tions with Ulex europaeus I. is a more sensitive technique for detecting intravascular tumor invasion." Also, some melanoma cells are PAS-positive (melanoma cells may contain a heavily glycosylated glycoprotein antigen'"). Nevertheless, the PAS without hematoxylin stain should be available in every laboratory; the application of confocal laser scanning microscopy is now restricted to research laboratories. The distribution of various vascular patterns is also important to those who treat ciliary body and choroidal melanomas: networks are particularly important in establishing a microenvironment of oxygen and nutrients within a tumor." Radiation therapy often targets the tumor vasculature. 50 The response to hyperthermia may be related to tumor vascularity." Perfusion with chemotherapeutic agents or tumor-specific antibodies'? depends on the microcirculation. The distribution of tumor vessel patterns in uveal melanomas is very heterogeneous'"; this observation has recently been cited as a possible cause of "inhomogeneous tumor regression" in uveal melanomas treated with ruthenium plaques.53 In our experience, it is unusual for a tumor to contain only networks. Usually, tumors contain a combination of vascular patterns; nearly half the tumors in this study contained areas that were not vascularized (zones of "silence"). We only have limited information concerning what causes the shape of these various vascular patterns. In vitro studies suggest that the first vascular supply of tumors arises either from normal vessels at the periphery" or from normal vessels that may become incorporated into the mass.55 This observation might explain the origin of the vascular pattern that we described as "normal": tumor cells growing around normal vessels. Coincidentally, the "normal" vascular pattern is the only pattern illustrated in photomicrographs of choroidal nevi in animals56-58 and humans.59 Normal vessels may be compressed by tissue pressure/" become occluded or even disintegrated.?' perhaps giving rise to the pattern that we designated as "silent." Perhaps other growth patterns form because of the cell types of the tumor cells55,62: although there is a relationship be-
Folberg et al . Uveal Melanoma Tumor Vessels tween cell type and certain vascular patterns, it does not hold, for example, that straight vessels always develop in zones of spindle melanoma cells or that loops and networks form in zones of epithelioid cells." Perhaps the extracellular matrix controls the shapes of these vascular patterns. 6 3 ,64 These vascular patterns may not be unique to uveal melanomas. The development of networks has been described in experimental tumor systems." Although not necessarily identified as such, we have found illustrations of avascular zones, arcs with and without branching, loops, and networks in other tumors such as adenocarcinoma of the breast 2 1•23 and colon/?
References I. Seddon JM, Albert OM, Lavin PT, Robinson N. A prognostic factor study of disease-free interval and survival following enucleation for uveal melanoma. Arch Ophthalmol 1983;101: 1894-9. 2. Sham mas HF, Blodi FC. Prognostic factors in choroidal and ciliary body melanomas. Arch Ophthalmol 1977;95: 63-9. 3. McLean IW, Foster WO, Zimmerman LE. Uveal melanoma: location , size, cell type, and enucleation as risk factors in metastasis. Hum Pathol 1982;13:123-32. 4. Seddon JM , Gragoudas ES, Albert OM, et al. Comparison of survival rates for patients with uveal melanoma after treatment with proton beam irradiation or enucleation. Am J Ophthalmol 1985;99:282-90. 5. Shammas HF, Blodi Fe. Orbital extension of choroidal and ciliary body melanomas. Arch Ophthalmol 1977;95:2002-
5.
6. Callender GR. Malignant melanotic tumors of the eye: a study of histologic types in III cases. Trans Am Acad Ophthalmol Otolaryngol 1931;36: 131-42. 7. McLean IW, Foster WD, Zimmerman LE, Gamel JW. Modifications of Callender's classification of uveal melanoma at the Armed Forces Institute of Pathology. Am J Ophthalmol 1983;96:502-9. 8. Gamel JW, McLean I, Greenberg RA, et al. Objective assessment of the malignant potential of intraocular melanomas with standard microslides stained with hematoxylineosin. Hum Pathol 1985;16:689-92. 9. Huntington A, Haugan P, Gamel J, McLean 1. A simple cytologic method for predicting the malignant potential of intraocular melanoma. Pathol Res Pract 1989;185:631-4. 10. McCurdy J, Gamel J, McLean 1. A simple, efficient, and reproducible method for estimating the malignant potential of uveal melanoma from routine H & E slides. Pathol Res Pract 1991;187:1025-27. II. Gamel JW, McCurdy 18, McLean IW. A comparison of prognostic covariates for uveal melanoma. Invest Ophthalmol Vis Sci 1992;33:1919-22. 12. Augsburger JJ , Shields JA, Folberg R, et al. Fine needle aspiration biopsy in the diagnosis of intraocular cancer. Cytologic-histologic correlations. Ophthalmology 1985;92:3949. 13. Folberg R, Augsburger JJ, Gamel JW, et al. Fine-needle aspirates of uveal melanomas and prognosis. Am J Ophthalmol 1985;100:654-7. 14. Char OH, Kroll SM, Stoloff A, et al. Cytomorphometry of uveal melanoma. Comparison of fine needle aspiration bi-
opsy samples with histologic sections. Anal Quant Cytol Histol 1991;13:293-9. IS. Srivastava A, Hughes LE, Woodcock JP , Laidler P. Vascularity in cutaneous melanoma detected by Doppler sonography and histology: correlation with tumour behaviour. Br J Cancer 1989;59:89-91 . 16. Srivastava A, Laidler P, Hughes LE, et al. Neovascularization in human cutaneous melanoma: a quantitative morphological and Doppler ultrasound study. Eur J Cancer Clin On col 1986;22:1205-9. 17. Srivastava A, Hughes LE, Woodcock JP, Shedden EJ. The significance of blood flow in cutaneous malignant melanoma demonstrated by Doppler flowmetry, Eur J Surg Oncol 1986;12:13-18. 18. Fallowfield ME, Cook MG. The vascularity of primary cutaneous melanoma. J Pathol 1991;164:241-4. 19. Camochan P, Briggs JC, Westbury G, Davies AJS. The vascularity of cutaneous melanoma: a quantitative histological study of lesions 0.85-1.25 mm in thickness. Br J Cancer 1991;64:102-7. 20. Barnhill RL, Fandrey K, Levy MA, et al. Angiogenesis and tumor progression of melanoma. Quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma. Lab Invest 1992;67:331-7. 21. Less JR , Skalak TC , Sevick EM, Jain RK . Microvascular architecture in a mammary carcinoma: branching patterns and vessel dimensions. Cancer Res 1991;51:265-73. 22. 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. 23. Bosari S, Lee AKC, DeLellis RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. Hum PathoI1992;23:755-61. 24. Macchiarini P, Fontanini G, Hardin MJ, et al. Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet 1992;340:145-6. 25. Bigler S, Deering RE, Brawer MK. Comparison of microscopic vascularity in benign and malignant prostate tissue. Hum Pathol 1993;24:220-26. 26. Coleman OJ, Lizzi FL. Computerized ultrasonic tissue characterization of ocular tumors. Am J Ophthalmol 1983;96:165-75. 27. Coleman OJ, Silverman RH, Rondeau MJ, et al. Correlations of acoustic tissue typing of malignant melanoma and histopathologic features as a predictor of death. Am J Ophthalmol 1990;110:380-8. 28. Miller MV, Herdson PB, Hitchcock Gc. Malignant melanoma of the uveal tract-a review of the Auckland experience. Pathology 1985;17:281-4. 29. Folberg R, Pe'er J, Gruman LM, et al. The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: a matched case-control stud y. Hum Pathol 1992;23:1298-1305. 30. Fine SL. How should we manage a patient with uveal melanoma [editorial]? Arch Ophthalmol 1985;103:910-1 I. 31. Folberg R, Gamel JW, Greenberg RA, et al. Comparison of direct and microslide pathology measurements of uveal melanomas. Invest Ophthalmol Vis Sci 1985;26:1788-91 . 32. de la Cruz PO Jr, Specht CS, McLean IW. Lymphocytic infiltration in uveal malignant melanoma. Cancer 1990;65: 112-15. 33. Terry TL, Johns JP . Uveal sarcoma-malignant melanoma. A statistical study of ninety-four cases. Am J Ophthalmol 1935;18:903-13. 34. Fleiss JL. Statistical Methods for Rates and Proportions, 2nd ed. New York: Wiley & Sons, 1981.
1397
Ophthalmology
Volume 100, Number 9, September 1993
35. Fuchs U, Kivela T, Summanen P, et al. An immunohistochemical and prognostic analysis of cytokeratin expression in malignant uveal melanoma. Am J PathoI1992;141:16981. 36. Kalbfleisch JD, Prentice RL. The Statistical Analysis of Failure Time Data. New York: Wiley & Sons, 1980. 37. Cox DR. Regression models and life-tables. J Roy Stat Soc [Ser. B] 1972;34:187-220. 38. SAS/ST AT Software: Changes and Enhancements. Cory, NC: SAS Institute, 1992. (SAS technical report; P-229.) 39. Zimmerman LE. Malignant melanoma of the uveal tract. In: Spencer WH, ed. Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Vol. 3. Philadelphia: WB Saunders, 1986;2072-139. 40. Jensen OA. Malignant melanomas of the uvea in Denmark 1943-1952. A clinical, histopathological, and prognostic study. Acta Ophthalmol (Suppl) 1963;75:141-89. 41. Coleman DJ, Lizzi FL, Silverman RH, et al. A model for acoustic characterization of intraocular tumors. Invest Ophthalmol Vis Sci 1985;26:545-50. 42. Wolff-Kormann PG, Kormann BA, Riedel KG, et al. Quantitative color Doppler imaging in untreated and irradiated choroidal melanoma. Invest Ophthalmol Vis Sci 1992;33: 1928-33. 43. Langer R. Angiogenesis-biomedical technology. In: Steiner R, Weisz PB, Langer R, eds. Angiogenesis: Key PrinciplesScience-Technology-Medicine. Basel: Birkhauser Verlag, 1992;23-5. 44. Holthofer H, Virtanen I, Kariniemi A-L, et al. Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest 1982;47:60-6. 45. Warren BA. The ultrastructure of the microcirculation at the advancing edge of Walker 256 carcinoma. Microvasc Res 1970;2:443-53. 46. Zinn KM, Stein/Pokorny K, Jakobiec FA, et al. Protonbeam irradiated epithelioid cell melanoma of the ciliary body. Ophthalmology 1981;88:1315-21. 47. Paku S, Paweletz N. First steps of tumor-related angiogenesis. Lab Invest 1991;65:334-46. 48. Folberg R, Donoso LA, Atkinson BF, et al. An antimelanoma monoclonal antibody and the histopathology of uveal melanomas. Arch Ophthalmol 1985;103:275-9. 49. Gaehtgens P. Why networks? Int J Microcirc Clin Exp 1992;11:123-32. 50. Hilmas DE, Gillette EL. Morphometric analyses of the microvasculature of tumors during growth and after x-irradiation. Cancer 1974;33: 103-10. 51. Braakman R, van der Valk P, von Delft JL, et al. The effects of ultrasonically induced hyperthermia on experimental tu-
1398
52. 53.
54. 55. 56.
57.
58. 59.
60. 61.
62. 63. 64.
65.
mors in the rabbit eye. Invest Ophthalmol Vis Sci 1989;30: 835-44. Jain RK. Delivery of novel therapeutic agents in tumors: physiological barriers and strategies. J Natl Cancer Inst 1989;81:570-6. Messmer E, Bornfeld N, Foerster M, et al. Histopathologic findings in eyes treated with a ruthenium plaque for uveal melanoma. Graefes Arch Clin Exp Ophthalmol 1992;230: 391-6. Skinner SA, Tutton PJM, O'Brien PE. Microvascular architecture of experimental colon tumors in the rat. Cancer Res 1990;50:2411-17. Vaupel P, Gabbert H. Evidence for and against a tumor type-specific vascularity. Strahlenther Onkol 1986;162:6338. Weisse I, Frese K, Meyer D. Benign melanoma of the choroid in a beagle: ophthalmological, light and electron microscopical investigations. Vet Pathoi 1985;22: 586-91. Folberg R, Baron J, Reeves RD, et al. Primary melanocytic lesions of the rabbit choroid following topical application of 7, 12-dimethylbenz[a]-anthracene: preliminary observations. J Toxicol Cutaneous Ocul Toxicol 1990;9:313-34. Pe'er J, Folberg R, Massicotte SJ, et al. Clinicopathologic spectrum of primary uveal melanocytic lesions in an animal model. Ophthalmology 1992;99:977-86. Naumann G, YanoffM, Zimmerman LE. Histogenesis of malignant melanomas of the uvea. I. Histopathologic characteristics of nevi of the choroid and ciliary body. Arch Ophthalmol 1966;76:784-96. Folkman J. Tumor angiogenesis. Adv Cancer Res 1985;43: 175-203. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989;49: 6449-65. Neetens A. Microcirculation in uveal melanomata. Bibl Anat 1977;15:297-300. Madri JA, Marx M. Matrix composition, organization and soluble factors: modulators of microvascular cell differentiation in vitro. Kidney Int 1992;41:560-5. Vernon RB, Angello JC, Iruela-Arispe ML, et al. Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab Invest 1992;66:536-47. Sun X-F, Zhang H, Wu X-C, et al. Microvascular corrosion casting of normal tissue, transitional mucosa and adenocarcinoma in the human colon. Acta Oncol 1992;31:3740.
Background: It is possible to identify at least nine vascular patterns in melanomas of the ciliary body and choroid from histologic sections. An association between the presence of at least one closed vascular loop and death from metastases was shown in a matched-pair, case-control study of 40 patients whose eyes were removed for ciliary body or choroidal melanomas. Methods: Two independent observers who were masked to the follow-up of patients examined histologic preparations of 234 eyes removed for ciliary body or choroidal melanomas for the presence of each of the tumor vascular patterns. Statistical analyses included tests for interobserver reliability, Kaplan-Meier survival curves, and the fitting of Cox regression models. Results: The detection of each of the nine vascular patterns is highly reproducible. The Cox model indicates that the presence of vascular networks, defined as at least three back-to-back closed vascular loops, is the feature most strongly associated with death from metastatic melanoma. Other significant factors in the Cox model include (in descending order of importance) largest tumor dimension, mitoses, the parallel with cross-linking vascular pattern, age, the presence of tumor-infiltrating lymphocytes, and male gender. Conclusions: The presence of vascular networks provides the most significant association with death from metastatic melanoma of all variables tested. The presence of this pattern should be recorded on pathology reports. If it becomes possible to detect this vascular pattern clinically using a noninvasive imaging technique, then ophthalmologists may be able to determine the likely biologic behavior of a melanoma before resorting to the removal of tissue. Ophthalmology 1993;100:1389-1398
The treatment of most cancers is based on a histologic assessment of the tumor's biologic behavior (grade) and the extent of spread from the primary site (stage).
Ophthalmologists must infer the biologic tumor grade from clinical observations: the largest tumor dimension (LTD) (especially the largest diameter in contact with the
Originally received: January 12, 1993. Revision accepted: March 8, 1993.
Dr. Parys Van-Ginderdeuren is currently at the Universitaire Ziekenhuizen St.-Rafael, Leuven, Belgium. Supported by National Institutes of Health grant EY07043, Bethesda, Maryland, and in part from an unrestricted grant from Research to Prevent Blindness, Inc, New York, New York. Dr. Rummelt is the recipient of grant Ru464/3-1 from the Deutsche Forschungsgemeinschaft, Germany. Reprint requests to Robert Folberg, MD, University of Iowa, 100 Medical Research Center, Rm 233, Iowa City, IA 52242-1182.
I
Department of Ophthalmology, University of Iowa, Iowa City.
2
Department of Pathology, University of Iowa, Iowa City.
Division of Biostatistics of the Department of Preventive Medicine, University of Iowa, Iowa City. 3
Department of Ophthalmology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
4
1389
Ophthalmology
Volume 100, Number 9, September 1993
Figure 1. The nine morphologic vascular patterns of primary ciliary body and choroidal melanomas (all photomicrographs: periodic acid-Schiff without hematoxylin; original magnification, X280). Top left, normal: tumor cells grow around normal choroidal vessels that are either filled with blood (n) or vacant (N). Top right, silent: no normal vessels or new vessels are identified. Center left, straight pattern: a straight vessel connects with a normal vessel (N). Center right, parallel: straight vessels are oriented in a parallel array. Bottom left, parallel with cross-linking: the cross-links are identified by arrowheads. Bottom right, arcs: arcs are fragments of curved vessels or incompletely closed loops. (Fig 1 continues)
sclera), I the location of the tumor (tumors situated anteriorly are more associated with subsequent metastases than those located posteriorlyj.i" and the presence or absence of extraocular extension' as detected by ultraso-
1390
nography. After an eye has been removed, pathologists may assign a true histologic grade (i.e., the likelihood of a patient dying of metastatic melanoma) by considering the following features: cell type,6,7 cytomorphometric as-
Folberg et al
Uveal Melanoma Tumor Vessels
Figure 1, continued. Top, arcs with branching: dichotomous branching from fragments of incompletely closed loops. Center, loops: an island of tumor is surrounded by a large, closed vascular loop (L) which is to the right of a vacant, normal vessel (N). Bottom, networks: back-to-back adjacent loops. Notice the relationship of the network to a normal vessel (N).
sessments of the nucleoli.r '? and nucleolar measurements combined with the largest area of scleral contact!' (along with those features that are also detectable clinically: tu-
mor size, location, and presence of extraocular extension). Although fine-needle aspiration biopsies of uveal masses may distinguish between primary melanomas and tumors metastatic to the uvea, these specimens do not accurately reflect the cell type identified from histologic sections." Also, there is no association between cytomorphometric analyses of nucleolar dimensions measured from fineneedle aspiration biopsy specimens and sections from eyes that have been removed. 13,14 Therefore, the management of uveal melanomas would be enhanced if a noninvasive method were available to grade the likelihood of a tumor generating metastases. Recently, the dermatologic community developed noninvasive techniques for grading cutaneous melanoma, Using ultrasonographic techniques, it is possible to detect tumor vascularity in cutaneous melanomas15 that correlates well with corresponding histologic sections.l'"!" In addition, there is an association between the density of tumor vessels and prognosis in some cutaneous melanomas. 15,18-20 Similar relationships between blood vessel counts and prognosis have been established for adenocarcinomas of the breast,21-23 non-small cell carcinoma of the lung," and carcinomas of the prostate." The ophthalmic community also has been interested in the ultrasonic detection oftumor vascularity. Coleman and co-workers" correlated acoustic patterns with the Callender cell type, but they did not discover an association between vascularity and cell type." Miller and coworkers'" reported an association between the vascularity of uveal melanomas on tissue sections and prognosis, but this relationship has not been confirmed (K. Egan, personal communication; May 1992). Carnochan and associates 19 discovered that the density of tumor vessels did not correlate well with survival for cutaneous melanomas measuring between 0.85 and 1.25 mm. These investigators suggested that "the assessment of other features of the tumour vasculature may provide a useful complement to simple morphometry." Instead of measuring tumor vessel density, Folberg and coworkers" identified nine vascular patterns in tissue sections of uveal melanomas; in a matched-pair case-control study of 40 eyes, the vascular pattern most closely associated with death from metastatic melanoma after enucleation was the presence of one or more closed vascular loops. The presence of these loops also was associated with mitotic figures and epithelioid cells, histologic features associated with an unfavorable outcome after enucleation. We now report the relationships between tumor vascular patterns and survival in a larger series of eyes removed for uveal melanoma.
Materials and Methods We asked the Tumor Registry of the University of Iowa to identify all patients who had a diagnosis of "intraocular melanoma." Of the 7 80 cases identified, 199 did not have any pathologic material on file in our laboratory. Because we needed to use special stains to demonstrate the microcirculation of these melanomas, we required the use
1391
Ophthalmology
Volume 100, Number 9, September 1993
of paraffin block to recut and stain slides according to the methods described below. We could not locate the blocks of 200 cases. An additional 147 cases were eliminated for one or more ofthe following reasons: the blocks that were available did not contain complete sections of tumor that were representative in size compared with the dimensions recorded in the gross pathology section of the pathology report, pure iris melanomas, eyes that had received any form of pre-enucleation treatment such as radiation, eyes from patients identified as having more than one cancer (including bilateral uveal melanoma), eyes from patients enrolled in the Collaborative Ocular Melanoma Study." nevi, and tumors more than 50% necrotic. Thus, 234 tumors met the criteria for entry into this study. The Iowa Tumor Registry provided follow-up data for all cases. At least two adjacent paraffin sections were cut at 5 to 6 Jlm. One section was stained with hematoxylin-eosin and was treated by permanganate bleaching if nuclear features were obscured by melanin. One slide was stained with periodic acid-Schiff (PAS) without hematoxylin counterstaining (with permanganate bleaching in cases of heavily pigmented tumors) and was viewed with a green filter; the accuracy of this technique compares favorably with more specific methods of identifying vessels in the uveal melanoma microcirculation." One of us (RF) recorded the following information from the hematoxylin-eosin slide: the tumor location, the largest tumor dimension (there is no significant difference between using tumor dimension measurements from glass slides and gross pathology reports in studies of patient survival:"), the number of mitotic figures per 40 highpower fields (X40 objective using a X10 eyepiece), the cell type using the modified Callender classification," the presence of 100 lymphocytes per 20 high-power fields." and the presence of extraocular extension or a diffuse growth pattern. Two of us (VR and RPV) who were not involved in the initial description of the nine morphologic patterns of tumor vessels in uveal melanoma" independently reviewed the slides stained with PAS without hematoxylin and recorded the presence or absence of each of the following nine vascular patterns" (Fig 1): 1. Normal Pattern: Tumor cells grow around normal
2. 3. 4. 5.
1392
choroidal vessels without compressing them. Three normal vessels must be detected beneath Bruch's membrane at Xl 0 magnification with a X10 ocular for this pattern to be recorded as present (dilated normal vessels seen above Bruch's membrane have been attributed to stagnant venous return"), Silence: No normal or new tumor vessels are present in a Xl 0 field viewed with a X10 ocular. Straight Vessels: Randomly distributed straight vessels lack dichotomous branching and are not cross-linked to each other. Parallel Pattern: Straight vessels, lacking dichotomous branching or cross-linking, are aligned parallel to each other. Parallelwith Cross-linking: Parallel vessels are crosslinked to each other; this pattern may resemble switching tracks in a rail yard.
6. Arcs: Arcs are incomplete loops. We did not obtain serial sections through the tumor to discover if arcs became loops in deeper sections. 7. Arcs with Branching: Arcs show dichotomous branching. 8. Loops: A loop is completely closed. The presence of one loop is sufficient to record this pattern as present. 9. Networks: A network is composed of least three back-to-back closed loops. By definition, if networks are present, loops are present. None of the investigators examining the slides knew the follow-up status. The investigators recorded the presence or absence of each ofthe nine vascular patterns from the slides stained by the PAS without hematoxylin method independently; the observations for each pattern in each case were compared for interobserver reliability. Discrepancies between the two observers were resolved by consensus, and this consensus observation was used in further statistical analyses. We tested the interobserver reliability for the identification of each vascular pattern using the chance-corrected measure of agreement (Kappa statistic). 34 The continuityadjusted chi-square test was used when appropriate to explore possible associations between individual vascular patterns and the following features derived from the histologic examination: tumor location, cell type, mitoses, the presence of more than 100 lymphocytes per 20 highpower fields, extraocular extension, and a diffuse growth pattern. We tested for an association between each vascular pattern and location with three types ofcomparisons: (1) all tumors confined to the choroid versus tumors having a component in the ciliary body, (2) tumors in the choroid posteriorly versus tumors in the choroid anteriorly versus tumors involving the ciliary body, and (3) tumors of the choroid posteriorly with tumors of the choroid anteriorly. We tested the possible association between each vascular pattern and cell type in two ways: (1) by using three categories of the modified Callender classification7 (spindle cell melanoma, mixed cell type and epithelioid), and (2) the presence or absence of any epithelioid cells (spindle cell melanoma versus a combination of mixed cell type and epithelioid tumors). 35 The primary outcome variables for individuals whose eyes are included in this study consist of (I) the time to death from metastatic melanoma or from other causes, or (2) the time to follow-up for those patients who are still alive. The analysis described here focuses on the time to death from metastatic melanoma. We treated time-todeath from other causes, time-to-follow-up for living patients, and time to last contact for patients reported as "lost to follow-up" as censored times in the data analyses. The primary statistical model fit is the Cox proportional hazards model. This model is appropriate for the analysis of failure time or mortality data and is sufficiently flexible to handle multiple covariables. The primary assumption involved in this model is that of proportional hazards, namely the hazard function for an individual with one set of characteristics is proportional to the hazard function
Folberg et al . Uveal Melanoma Tumor Vessels Table 1. Agreement between Two Independent Observers for Each Vascular Pattern
Pattern
% Agreement
Normal Silence Straight Parallel Parallel with cross-linking Arcs Arcs with branching Loops Networks
94 91 92 83
77 81 83
84 80
Chance Corrected Measure of Agreement (kappa statistic]"
-0.028 0.807 0.056 0.479 0.530
0.444 0.611 0.667 0.591
• p < 0.0001 for all observations except the normal pattern. In this pattern, nearly every observation is in one category so there is little variability; the % agreement may be a more meaningful statistic for this variable.
for an individual with another set of characteristics. This ratio is commonly thought of as the relative risk. Stepwise regression techniques were used to fit several models involving the selection of variables; score chisquare statistics" with significance levels of 0.05 were used for entry and removal in the stepwise model building. We fit survival curves for individual groups using the KaplanMeier method." We calculated the log-likelihood ratio statistic (-210gL) to test several regression coefficients simultaneously and to compare competing models. Wald chi-square" was used to assess final model coefficients. In all instances, the analyses involved fitting Cox regression models." We used the SAS PH REG procedure."
Results The tumors came from 106 men (45.3%) and 128 women (54.7%).The agesof patients at the time of diagnosis ranged Table 2. Frequency Distributions for Vascular Patterns'" in Choroidal and Ciliary Body Melanomas Cases Identified (n = 234) Vascular Pattern
No. (0/0)
Normal Silent Straight Parallel Parallel with cross-links Arcs Arcs with branching Loops Networks
226 (97) 104(44)
226 (97) 198(85) 140(60) 182 (78) 159 (68) 131 (56) 106(45)
from 18 to 88 years (median, 59 years). Follow-up ranged from 6 months to 39.7 years (median survival, 7.6 years). Follow-up information showed that 101 patients (43.2%) were alive without any evidence of cancer, 51 (21.8%) had died without evidence of metastasis, 67 (28.6%) had died of metastatic melanoma, and 15 (6.4%)were lost to followup. The LTD in contact with the sclera" ranged from 5 to 23 mm (median, 12 mm). Sixty-seven tumors (28.6%) were small « 10 mm in diameter), 117 tumors (50%) were medium-sized (> 10 mm but < 15 mm in diameter), and 50 tumors (21.4%) were large (~15 mm in diameterj." One hundred thirty-two tumors (56.4%).were located in the choroid posterior to the equator, 48 (20.5%) were located in the choroid anterior to the equator without involvement of the ciliary body, 47 (20.1%) involved the choroid and ciliary body, 2 (0.9%)involved only the ciliary body, and 5 (2.1%) involved the choroid, ciliary body, and iris. One hundred twelve tumors (47.9%) were classified as spindle cell melanomas, 103 (44%)were classifiedas mixed cell type, and 19 were classified as epithelioid melanomas (8.1%).7 At least one mitotic figure was identified in 112 tumors (47.9%). At least 100 lymphocytes per 20 highpower fields'? were identified in 67 tumors (28.6%). Extraocular extension was identified in nine tumors (3.8%), and seven (3%) were classified as diffuse. The identification of each vascular pattern was highly reproducible (Table 1). Table 2 summarizes the frequency distribution for each vascular pattern. Parallel with crosslinking, arcs, arcs with branching, loops, and networks are more likely to be detected in the ciliary body than in the choroid; parallel with cross-linking, arcs, arcs with branching, loops, and networks are more likely to be detected in the choroid anterior to the equator than in the choroid posteriorly. Chi-square tests showed that no vascular pattern had a significant association with extraocular extension or a diffuse growth pattern. A statistically significant association was identified between the presence of epithelioid cells" (spindle cell melanomas versus mixed and epithe-
1393
Ophthalmology
Volume 100, Number 9, September 1993
lioid cell melanomas) and the following vascular patterns: parallel (P = 0.001), and arcs, parallel with cross-linking, arcs with branching, loops, and networks (all P < 0.000 I). The absence of epithelioid cells was associated with the silence pattern (P = 0.007). The presence of at least 100 tumor-infiltrating lymphocytes per 20 high-power fields was associated with the following vascular patterns: parallel with cross-linking (P = 0.001), loops (P = 0.004), and networks (P = 0.00 I). Figure 2 shows Kaplan-Meier survival curves generated from deaths due to metastatic melanoma according to location, LTD, cell type, parallel with cross-linking, loops, and networks. Briefly, at the lO-year follow-up visit, the survival of patients whose tumors lack the vascular patterns of parallel with cross-linking, loops, and networks is 91.7%, 91.1 %, and 88.3%, respectively, versus 56.9%, 55.4%, and 50.7% if these patterns are present. KaplanMeier survival curves for all patient deaths are similar to those illustrated for deaths from metastatic melanoma. When considering all variables, including the vascular patterns, the complete model (i.e., the one that includes all variates) suggested that statistical collinearity exists between two of the vascular patterns. These patterns (arcs and arcs with branching) were dropped from the analysis. We initiated the stepwise regression after these variables were deleted. Table 3 summarizes the results from fitting the stepwise regression procedure and includes a summary of the stepwise modeling at each step and the individual chi-square statistic from the score procedure. The most important variable (and the one to enter the model first) is the network pattern (chi-square = 40.84; P = 0.0001). At the second step, the next most important variable, given that the network pattern was in the model, was LTD (chisquare = 14.64; P = 0.0001). Table 3 is self-explanatory thereafter. None of the variables entered in the preceding steps was removed (i.e., each retained its importance in the model). After the seventh step, no additional variables reached significance for entry into the model. Therefore, the final model included networks, LTD, mitoses, parallel with cross-linking, age, tumorinfiltrating lymphocytes, and the patient's sex (male gender). Table 4 summarizes the estimated Cox model regression coefficients. Note that each estimate is a positive number, indicating that the presence of networks, at least one mitotic figure and the parallel with cross-linking patterns are associated with a higher risk of death from metastatic melanoma. Table 4 also includes standard errors along with chi-square statistics. Each of these partial chisquare statistics has a very small P value, indicating the importance of these terms in the model. Table 4 also includes the value of the log likelihood with its seven degrees of freedom. This stepwise modeling suggested that these seven variables were the most important; of these seven, the two most important variables were the presence of vascular networks and the LTD.
Discussion In a matched-pair case-control study of 40 eyes removed for ciliary body or choroidal melanoma, we showed that
1394
the presence of at least one closed vascular loop was strongly associated with death from metastatic melanoma." Our current study confirms that vascular patterns are more strongly associated with death from melanoma after enucleation than the LTD, location, cell type, mitotic count, and tumor-infiltrating lymphocytes. In the Cox model considering deaths from melanomas, the presence of networks is the factor most strongly associated with death from metastatic melanoma. In the Cox model considering all deaths, the presence of networks is also the most significant variable. Parallel with cross-linking vessels may be a variant of networks: if vessels cross-link at more than one place, they form elongated loops. Ifthese stretched-out loops are arranged back to back, then they form elongated networks. It is not surprising that this multivariate analysis offactors shows networks and parallel with cross-linking to be more significant than loops, the factor identified as most significant in our univariate analysis": networks are composed of loops. By examining tissue sections from glass slides, we have only sampled a two-dimensional plane. There is a heterogeneous distribution of these patterns in two dimensions (i.e., when viewed on the surface of a glass slide), so it is reasonable to expect a heterogeneous distribution of these patterns deeper in the tissue block." Not only may some patterns be present or absent in deeper planes, it is possible that arcs and arcs with branching become loops when imaged in three dimensions: the detection of both arcs and arcs with branching in two-dimensional tissue sections is associated with death from metastatic melanomas in univariate analyses but is not significant in multivariate analysis. It is important to realize that tumor vessels develop in three dimensions. In some studies, investigators merely counted the number of blood vessel cross-sections per unit area (vessel density) and linked these counts to outcome (i.e., breast cancer,21-23 some forms of cutaneous melanoma,15,18,19,28 and prostatic cancer"), It is quite possible that the same vessel is counted more than once because vessels may weave in and out of section planes. In addition, the technique of counting tumor vessel cross-sections may not even be applicable to uveal melanoma: how often would a vessel be counted if it were part of a vascular network of closed loops? It is noteworthy that cell type and tumor location, variables described previously as important associations with death from metastatic melanoma.v" did not appear in the stepwise modeling and in any step along the way. Networks have an effect beyond any that may exist due to cell type when both variables are considered in any statistical formulation. From any point of view for these data, the presence of networks is the more dominant characteristic and the one that accounts for the most statistical variation. Two of the variables that appear in our model, age and sex, are not often cited as risk factors for death from metastatic uveal melanoma. However, Jensen'" also observed that patients older than 40 years of age at the time of treatment had a worse prognosis than younger patients.
Folberg et al . Uveal Melanoma Tumor Vessels Z
9
t! Z
::;J
:.r..
1.0
',-
",
' \'l.\,
"L
0.8
::;J
._._ _ .~.M_. __.
-L - L -
0.6
~
f::2
=
p-vatu e
P::l
0.8
::;J
0.6
'\
\
·L __.
ell
..... ._.... _,
i.
0.4
p-value
2: > ~
0.2
0.2
:J
en 0.0
0.0 10
20
15
1.0
u
30
25
35
a
40
. _ --
Spindle
....... ...._.
Mixed
-
Epithelioid
-
-
0.8
1.0
:J
0.8
No Pa rallel Cross
---~
't..
'---1--,
p-value
p-vatua
0.4
.0002
i5
....l
>
0.4
:>
0.2
0.2
~
::;J
:J
en
en 0.0 0
10
15
20
30
J 25
35
0.0 0
40
10
15
TIME (YEARS)
1.0
~_ _......._
u
~
_
-
.
-
L._.•._m._ _
1
No LOOP --
__.__ _ _
With loop
I
Z
6z
_ __••...__..__.. ..
0.8
~
::;J
p-value
0.6
§
i5
40
~
~--- l
0.8
---
·L
..
.
NoNo~
With NetwOrk
..._.._.._..__...
I ._
0.6
p-value
.000 1
0.4
....l
~
0.2
0.2
~
:J
::;J
en
35
~
~ 0
0.4
....l
::= > ~
1-
1.0
E9
~
E-
30
Z
.0001
E9
en
25
20
TIME (YEARS)
Z
0 1==
.0001
P::l
~ en
Z
_ ._---- - -- _.... _----- _.
0.6
:J
....l
Z 0 1==
Wi th Parallel Cross
----I
1==
0.6
::=
> ~
40
35
~
~
s
30
25
Z 0
E-
en
Z 0
C Z
Z
E9
20
15
10
TIME (YEARS)
0
§
.000 1
TIME (YEARS)
it
._._.._
_.•
....l
0
z
.._..__....
._._....-.... ,,
i5
::;J
Z 0 1==
L.T.D. ( 10-15) L.T D. ( > 15 )
, _ _ .__ ,
E0.4
en
it E9 ~
.0004
Cl ....l
::= > ~
1.
Z 0 1==
_
L.T.D . ( <= 10 )
1.0
C z
Ciliary Body -'~-'"\.t...-..,
Z
0 1==
Z 0
Choroid
ell
0.0 0
10
15
20
25
TIME (YEARS )
30
35
40
0.0 0
10
15
20
25
30
35
40
TIME (YEARS)
Figure 2. Kaplan -Meier sur vival curves for deaths from metastatic melanoma. Top left, location : tumors involving th e ciliary bod y versus tum or s confined to the choroid. Top right, largest tumor dimen sion (L.T .D.): small melanomas « 10 mm) versus medium-sized melan om as (10- 15 mm ) versus large melan omas (> 15 mm). Center left, cell type: spindle cell melanomas versus melan om as of the mixed cell type versus epithelioid melanomas. Center right, melanomas without the parallel with cross-linking pattern versus tumors with this patt ern. Bott om left, melanomas with out loops versus melanomas with loops . Bottom right, melanomas without vascular network s versus melan omas with netw orks.
1395
Ophthalmology
Volume 100, Number 9, September 1993 Table 4. Regression Coefficients and Standard Errors*
Table 3. Summary of Stepwise Regression Procedures Variable
Step
Networks Largest tumor dimension Mitoses Parallel with cross-linking Age > 100 tumor-infiltrating lymphocytes/20 HPF Sex
1 2 3 4 5 6 7 HPF
=
P
Variable
Estimate
SE
Wald Chi-square
P
14.64 9.06 10.32 6.07 5.30
0.0001 0.0001 0.0026 0.0013 0.0137 0.0214
1.056 0.108 0.148 1.172 0.025 0.642
0.3076 0.0387 0.0375 0.4148 0.0095 0.2683
11.78 7.75 15.50 7.98 7.13 5.73
0.0006 0.0054 0.0001 0.0047 0.0076 0.0167
4.57
0.0172
Networks Largest tumor dimension Mitoses Parallel with cross-linking Age > 100 tumor-infiltrating lymphocytes/20 HPF Male gender
0.536
0.2533
4.48
0.0343
Score Chisquare
40.84
high power field.
Jensen'? also noted that the survival for women with uveal melanoma was more favorable than for men. It will be necessary in a future study to evaluate the effect of vascular patterns on survival when cytomorphometric measurements'"!" are entered into a multivariate model. However, even if cytomorphometric measurements prove to have a greater effect on mortality than the presence of networks, these measurements are only useful after the eye has been removed.P:'" On the other hand, if it is possible to develop a noninvasive technique to detect vascular networks or parallel with cross-linking patterns (by ultrasonic acoustic microscopy.P-":" Doppler ultrasonography.f or other imaging techniques':'), then it may be possible to assign a histologic tumor grade in vivo. We recommend that the presence or absence of these patterns be recorded on histopathology reports. Our current study suggests that the classification system of vascular pattern can be taught easily to others: the two investigators (VR and RPV) in this study who graded each tumor independently for the presence or absence of each vascular pattern did not participate in our pilot study." Each of these individuals mastered the identification of vascular patterns within I day. There is an extremely high degree of agreement between both of the observers, suggesting that the use of the vascular patterns as an indicator of the prognosis after enucleation is highly reproducible. In addition, the laboratory techniques for the detection of these vessels may be applied easily in any pathology laboratory. We previously showed a correspondence between the modified PAS (without hematoxylin) stain used in this study and the specific method of detecting tumor blood vesselsin uveal melanomas (using the confocal laser microscopy and fluorescent Ulex europaeus I., a lectin specific for the identification of vascular endotheliumr"; the uvea lacks lymphatics. A prominent basement membrane zone frequently surrounds tumor blood vessels,29,45-47 a histologic feature that accounts for positive staining of these vessels with PAS. There are some drawbacks to the simple technique of applying PAS without hematoxylin staining to uveal melanomas to detect tumor blood vessels. First, staining sec-
1396
.
= standarderror; HPF = high power field. -2logL = 674.079; df = 7; modelchi-square = 86.850.
SE
tions with Ulex europaeus I. is a more sensitive technique for detecting intravascular tumor invasion." Also, some melanoma cells are PAS-positive (melanoma cells may contain a heavily glycosylated glycoprotein antigen'"). Nevertheless, the PAS without hematoxylin stain should be available in every laboratory; the application of confocal laser scanning microscopy is now restricted to research laboratories. The distribution of various vascular patterns is also important to those who treat ciliary body and choroidal melanomas: networks are particularly important in establishing a microenvironment of oxygen and nutrients within a tumor." Radiation therapy often targets the tumor vasculature. 50 The response to hyperthermia may be related to tumor vascularity." Perfusion with chemotherapeutic agents or tumor-specific antibodies'? depends on the microcirculation. The distribution of tumor vessel patterns in uveal melanomas is very heterogeneous'"; this observation has recently been cited as a possible cause of "inhomogeneous tumor regression" in uveal melanomas treated with ruthenium plaques.53 In our experience, it is unusual for a tumor to contain only networks. Usually, tumors contain a combination of vascular patterns; nearly half the tumors in this study contained areas that were not vascularized (zones of "silence"). We only have limited information concerning what causes the shape of these various vascular patterns. In vitro studies suggest that the first vascular supply of tumors arises either from normal vessels at the periphery" or from normal vessels that may become incorporated into the mass.55 This observation might explain the origin of the vascular pattern that we described as "normal": tumor cells growing around normal vessels. Coincidentally, the "normal" vascular pattern is the only pattern illustrated in photomicrographs of choroidal nevi in animals56-58 and humans.59 Normal vessels may be compressed by tissue pressure/" become occluded or even disintegrated.?' perhaps giving rise to the pattern that we designated as "silent." Perhaps other growth patterns form because of the cell types of the tumor cells55,62: although there is a relationship be-
Folberg et al . Uveal Melanoma Tumor Vessels tween cell type and certain vascular patterns, it does not hold, for example, that straight vessels always develop in zones of spindle melanoma cells or that loops and networks form in zones of epithelioid cells." Perhaps the extracellular matrix controls the shapes of these vascular patterns. 6 3 ,64 These vascular patterns may not be unique to uveal melanomas. The development of networks has been described in experimental tumor systems." Although not necessarily identified as such, we have found illustrations of avascular zones, arcs with and without branching, loops, and networks in other tumors such as adenocarcinoma of the breast 2 1•23 and colon/?
References I. Seddon JM, Albert OM, Lavin PT, Robinson N. A prognostic factor study of disease-free interval and survival following enucleation for uveal melanoma. Arch Ophthalmol 1983;101: 1894-9. 2. Sham mas HF, Blodi FC. Prognostic factors in choroidal and ciliary body melanomas. Arch Ophthalmol 1977;95: 63-9. 3. McLean IW, Foster WO, Zimmerman LE. Uveal melanoma: location , size, cell type, and enucleation as risk factors in metastasis. Hum Pathol 1982;13:123-32. 4. Seddon JM , Gragoudas ES, Albert OM, et al. Comparison of survival rates for patients with uveal melanoma after treatment with proton beam irradiation or enucleation. Am J Ophthalmol 1985;99:282-90. 5. Shammas HF, Blodi Fe. Orbital extension of choroidal and ciliary body melanomas. Arch Ophthalmol 1977;95:2002-
5.
6. Callender GR. Malignant melanotic tumors of the eye: a study of histologic types in III cases. Trans Am Acad Ophthalmol Otolaryngol 1931;36: 131-42. 7. McLean IW, Foster WD, Zimmerman LE, Gamel JW. Modifications of Callender's classification of uveal melanoma at the Armed Forces Institute of Pathology. Am J Ophthalmol 1983;96:502-9. 8. Gamel JW, McLean I, Greenberg RA, et al. Objective assessment of the malignant potential of intraocular melanomas with standard microslides stained with hematoxylineosin. Hum Pathol 1985;16:689-92. 9. Huntington A, Haugan P, Gamel J, McLean 1. A simple cytologic method for predicting the malignant potential of intraocular melanoma. Pathol Res Pract 1989;185:631-4. 10. McCurdy J, Gamel J, McLean 1. A simple, efficient, and reproducible method for estimating the malignant potential of uveal melanoma from routine H & E slides. Pathol Res Pract 1991;187:1025-27. II. Gamel JW, McCurdy 18, McLean IW. A comparison of prognostic covariates for uveal melanoma. Invest Ophthalmol Vis Sci 1992;33:1919-22. 12. Augsburger JJ , Shields JA, Folberg R, et al. Fine needle aspiration biopsy in the diagnosis of intraocular cancer. Cytologic-histologic correlations. Ophthalmology 1985;92:3949. 13. Folberg R, Augsburger JJ, Gamel JW, et al. Fine-needle aspirates of uveal melanomas and prognosis. Am J Ophthalmol 1985;100:654-7. 14. Char OH, Kroll SM, Stoloff A, et al. Cytomorphometry of uveal melanoma. Comparison of fine needle aspiration bi-
opsy samples with histologic sections. Anal Quant Cytol Histol 1991;13:293-9. IS. Srivastava A, Hughes LE, Woodcock JP , Laidler P. Vascularity in cutaneous melanoma detected by Doppler sonography and histology: correlation with tumour behaviour. Br J Cancer 1989;59:89-91 . 16. Srivastava A, Laidler P, Hughes LE, et al. Neovascularization in human cutaneous melanoma: a quantitative morphological and Doppler ultrasound study. Eur J Cancer Clin On col 1986;22:1205-9. 17. Srivastava A, Hughes LE, Woodcock JP, Shedden EJ. The significance of blood flow in cutaneous malignant melanoma demonstrated by Doppler flowmetry, Eur J Surg Oncol 1986;12:13-18. 18. Fallowfield ME, Cook MG. The vascularity of primary cutaneous melanoma. J Pathol 1991;164:241-4. 19. Camochan P, Briggs JC, Westbury G, Davies AJS. The vascularity of cutaneous melanoma: a quantitative histological study of lesions 0.85-1.25 mm in thickness. Br J Cancer 1991;64:102-7. 20. Barnhill RL, Fandrey K, Levy MA, et al. Angiogenesis and tumor progression of melanoma. Quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma. Lab Invest 1992;67:331-7. 21. Less JR , Skalak TC , Sevick EM, Jain RK . Microvascular architecture in a mammary carcinoma: branching patterns and vessel dimensions. Cancer Res 1991;51:265-73. 22. 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. 23. Bosari S, Lee AKC, DeLellis RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. Hum PathoI1992;23:755-61. 24. Macchiarini P, Fontanini G, Hardin MJ, et al. Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet 1992;340:145-6. 25. Bigler S, Deering RE, Brawer MK. Comparison of microscopic vascularity in benign and malignant prostate tissue. Hum Pathol 1993;24:220-26. 26. Coleman OJ, Lizzi FL. Computerized ultrasonic tissue characterization of ocular tumors. Am J Ophthalmol 1983;96:165-75. 27. Coleman OJ, Silverman RH, Rondeau MJ, et al. Correlations of acoustic tissue typing of malignant melanoma and histopathologic features as a predictor of death. Am J Ophthalmol 1990;110:380-8. 28. Miller MV, Herdson PB, Hitchcock Gc. Malignant melanoma of the uveal tract-a review of the Auckland experience. Pathology 1985;17:281-4. 29. Folberg R, Pe'er J, Gruman LM, et al. The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: a matched case-control stud y. Hum Pathol 1992;23:1298-1305. 30. Fine SL. How should we manage a patient with uveal melanoma [editorial]? Arch Ophthalmol 1985;103:910-1 I. 31. Folberg R, Gamel JW, Greenberg RA, et al. Comparison of direct and microslide pathology measurements of uveal melanomas. Invest Ophthalmol Vis Sci 1985;26:1788-91 . 32. de la Cruz PO Jr, Specht CS, McLean IW. Lymphocytic infiltration in uveal malignant melanoma. Cancer 1990;65: 112-15. 33. Terry TL, Johns JP . Uveal sarcoma-malignant melanoma. A statistical study of ninety-four cases. Am J Ophthalmol 1935;18:903-13. 34. Fleiss JL. Statistical Methods for Rates and Proportions, 2nd ed. New York: Wiley & Sons, 1981.
1397
Ophthalmology
Volume 100, Number 9, September 1993
35. Fuchs U, Kivela T, Summanen P, et al. An immunohistochemical and prognostic analysis of cytokeratin expression in malignant uveal melanoma. Am J PathoI1992;141:16981. 36. Kalbfleisch JD, Prentice RL. The Statistical Analysis of Failure Time Data. New York: Wiley & Sons, 1980. 37. Cox DR. Regression models and life-tables. J Roy Stat Soc [Ser. B] 1972;34:187-220. 38. SAS/ST AT Software: Changes and Enhancements. Cory, NC: SAS Institute, 1992. (SAS technical report; P-229.) 39. Zimmerman LE. Malignant melanoma of the uveal tract. In: Spencer WH, ed. Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Vol. 3. Philadelphia: WB Saunders, 1986;2072-139. 40. Jensen OA. Malignant melanomas of the uvea in Denmark 1943-1952. A clinical, histopathological, and prognostic study. Acta Ophthalmol (Suppl) 1963;75:141-89. 41. Coleman DJ, Lizzi FL, Silverman RH, et al. A model for acoustic characterization of intraocular tumors. Invest Ophthalmol Vis Sci 1985;26:545-50. 42. Wolff-Kormann PG, Kormann BA, Riedel KG, et al. Quantitative color Doppler imaging in untreated and irradiated choroidal melanoma. Invest Ophthalmol Vis Sci 1992;33: 1928-33. 43. Langer R. Angiogenesis-biomedical technology. In: Steiner R, Weisz PB, Langer R, eds. Angiogenesis: Key PrinciplesScience-Technology-Medicine. Basel: Birkhauser Verlag, 1992;23-5. 44. Holthofer H, Virtanen I, Kariniemi A-L, et al. Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest 1982;47:60-6. 45. Warren BA. The ultrastructure of the microcirculation at the advancing edge of Walker 256 carcinoma. Microvasc Res 1970;2:443-53. 46. Zinn KM, Stein/Pokorny K, Jakobiec FA, et al. Protonbeam irradiated epithelioid cell melanoma of the ciliary body. Ophthalmology 1981;88:1315-21. 47. Paku S, Paweletz N. First steps of tumor-related angiogenesis. Lab Invest 1991;65:334-46. 48. Folberg R, Donoso LA, Atkinson BF, et al. An antimelanoma monoclonal antibody and the histopathology of uveal melanomas. Arch Ophthalmol 1985;103:275-9. 49. Gaehtgens P. Why networks? Int J Microcirc Clin Exp 1992;11:123-32. 50. Hilmas DE, Gillette EL. Morphometric analyses of the microvasculature of tumors during growth and after x-irradiation. Cancer 1974;33: 103-10. 51. Braakman R, van der Valk P, von Delft JL, et al. The effects of ultrasonically induced hyperthermia on experimental tu-
1398
52. 53.
54. 55. 56.
57.
58. 59.
60. 61.
62. 63. 64.
65.
mors in the rabbit eye. Invest Ophthalmol Vis Sci 1989;30: 835-44. Jain RK. Delivery of novel therapeutic agents in tumors: physiological barriers and strategies. J Natl Cancer Inst 1989;81:570-6. Messmer E, Bornfeld N, Foerster M, et al. Histopathologic findings in eyes treated with a ruthenium plaque for uveal melanoma. Graefes Arch Clin Exp Ophthalmol 1992;230: 391-6. Skinner SA, Tutton PJM, O'Brien PE. Microvascular architecture of experimental colon tumors in the rat. Cancer Res 1990;50:2411-17. Vaupel P, Gabbert H. Evidence for and against a tumor type-specific vascularity. Strahlenther Onkol 1986;162:6338. Weisse I, Frese K, Meyer D. Benign melanoma of the choroid in a beagle: ophthalmological, light and electron microscopical investigations. Vet Pathoi 1985;22: 586-91. Folberg R, Baron J, Reeves RD, et al. Primary melanocytic lesions of the rabbit choroid following topical application of 7, 12-dimethylbenz[a]-anthracene: preliminary observations. J Toxicol Cutaneous Ocul Toxicol 1990;9:313-34. Pe'er J, Folberg R, Massicotte SJ, et al. Clinicopathologic spectrum of primary uveal melanocytic lesions in an animal model. Ophthalmology 1992;99:977-86. Naumann G, YanoffM, Zimmerman LE. Histogenesis of malignant melanomas of the uvea. I. Histopathologic characteristics of nevi of the choroid and ciliary body. Arch Ophthalmol 1966;76:784-96. Folkman J. Tumor angiogenesis. Adv Cancer Res 1985;43: 175-203. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989;49: 6449-65. Neetens A. Microcirculation in uveal melanomata. Bibl Anat 1977;15:297-300. Madri JA, Marx M. Matrix composition, organization and soluble factors: modulators of microvascular cell differentiation in vitro. Kidney Int 1992;41:560-5. Vernon RB, Angello JC, Iruela-Arispe ML, et al. Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab Invest 1992;66:536-47. Sun X-F, Zhang H, Wu X-C, et al. Microvascular corrosion casting of normal tissue, transitional mucosa and adenocarcinoma in the human colon. Acta Oncol 1992;31:3740.