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Clinical Cancer Research Vol. 11, 73-78, January 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Expression of Vascular Endothelial Growth Factor in Uveal Melanoma Is Independent of 6p21-Region Copy Number

Mohamed H. Abdel-Rahman1,2, Elson L. Craig1, Frederick H. Davidorf1 and Charis Eng2

1 Department of Ophthalmology and 2 Clinical Cancer Genetics Program, Human Cancer Genetics Program, Comprehensive Cancer Center; Division of Human Genetics, Department of Internal Medicine, The Ohio State University, Columbus, Ohio

Requests for reprints: Frederick H. Davidorf, Department of Ophthalmology, Ohio State University, 456 West 10th Avenue, Suite 5B, Columbus, OH 43210. Phone: 614-293-8041; Fax: 614-293-6180; E-mail: Davidorf.1{at}osu.edu.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Overexpression of vascular endothelial growth factor (VEGF) and overrepresentation of the 6p region have been reported with a wide variation in uveal melanoma. The aim of the current study is to identify the frequency of copy number alteration in the 6p21 region and its correlation with the expression of VEGF in uveal melanoma.

Experimental Design: We studied 88 uveal melanomas for copy number change in the 6p region by comparative genomic hybridization and/or chromogenic in situ hybridization. Expression of VEGF protein was estimated by immunohistochemistry. In 15 tumors, VEGF mRNA expression was also studied by quantitative reverse transcription–PCR (RT-PCR) and VEGF splice variants were detected by RT-PCR.

Results: Copy number of the 6p21 region was successfully estimated in 37 tumors. In 10 (27%) of those, overrepresentation of the 6p21 region was detected. There was no statistically significant difference in VEGF expression between tumors with and without gain of 6p21 (P = 0.82). VEGF expression was not confined to the tumors and was also detected in the surrounding normal tissue. Expression of VEGF, detected by quantitative RT-PCR, was concordant with expression of VEGF protein. Different VEGF isoforms were expressed in different tumors with no obvious correlation with disease status.

Conclusion: VEGF is overexpressed in a significant number of uveal melanomas. It should be noted that VEGF is not a candidate oncogene in uveal melanoma with 6p gain/amplification. VEGF overexpression other than structural amplification is probably significant in the pathogenesis of uveal melanomas, and its mechanism must be sought.

Key Words: Molecular cytogenetics • comparative genomic hybridization (CGH) • Chromogenic in situ hybridization (CISH) • VEGF splice variants


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 
Uveal melanoma is the most common primary intraocular malignancy in adults. High tumor vascular density and abnormal vascular patterns have been reported as prognostic pathologic features indicating aggressive tumors (1, 2). Angiogenesis, the formation of new vessels, is a highly regulated process. There is strong evidence that vascular endothelial growth factor (VEGF) is a major regulator of both physiologic and pathologic angiogenesis (3). VEGF expression has been reported as a useful marker of tumor angiogenesis in a variety of tumors (4). Its expression correlates with tumor vascularity, metastasis, and tumor proliferation; thus, high expression of VEGF in tumor tissue may be an indication of poor prognosis (4).

VEGF, the gene encoding VEGF, is located on chromosomal region 6p21.1 and contains 8 exons. Analysis of VEGF cDNAs predicts five alternately spliced VEGF protein isoforms: VEGF189 (full length), VEGF206 (alternate splice site selection in exon 7 with insertion of 17 amino acids), VEGF 121(lacking exons 6 and 7), VEGF165 (lacking exon 6), and VEGF145 (lacking exon7; refs. 4, 5). VEGF isoforms differ in their expression patterns as well as their biochemical and biological properties (5). VEGF stimulates and induces migration and proliferation of endothelial cells and enhances vascular permeability and thrombogenicity (4, 5). Currently, there are several promising therapies available targeting the expression of VEGF in ocular diseases associated with florid angiogenesis (6–8).

There is a large variation in the frequency of VEGF expression in published studies of uveal melanomas. Kvanta et al. reported expression of VEGF in retinoblastomas but in none of the 10 uveal melanomas included in their study (9). In contrast, other investigators reported expression of VEGF in up to 94% of the uveal melanomas (10). More recent studies showed that although VEGF mRNA could be detected in all uveal melanomas by reverse transcription–PCR (RT-PCR), only 22% of tumors showed expression of VEGF protein (11).

Overrepresentation of the 6p region in uveal melanoma has been reported in several cytogenetic and molecular cytogenetic studies, with a frequency ranging between 31% and 57% (12–17). The smallest region of overrepresentation is located on 6p21-6pter, which includes the physical location of VEGF in 6p21 (14). As far as we know, there is no available report on the correlation between copy number change in the 6p region and the expression of VEGF in uveal melanoma. Therefore, we sought to identify the frequency of copy number alteration of the 6p21 region and its correlation with the expression of VEGF in uveal melanoma. Our results indicate that VEGF expression can be independent of the copy number ofVEGF.


   MATERIALS AND METHOD
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
RESULTS
 DISCUSSION
 REFERENCES
 
Samples. Specimens were obtained from the archives of the Departments of Ophthalmology and Pathology, Ohio State University. We studied 88 primary choroidal melanomas. Fresh-frozen tissues were available for 15 tumors, paraffin-embedded tissues were available for 84 tumors, and both paraffin-embedded and fresh-frozen tissues were available for 11 tumors. Sample collection was in accordance with an Institutional Review Board–approved protocol (2003C0057). Tissue microarrays were prepared from paraffin-embedded tissue as previously reported (18). A 1.5-mm representative core from each tumor tissue was included. For tumors with mixed morphology, at least two cores representing different morphologic regions were included.

RNA and DNA Extraction. RNA was extracted from fresh-frozen tumor tissues using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. DNA was extracted from fresh-frozen tumor tissues and from peripheral blood lymphocytes of healthy male and female volunteers using QIA amp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The integrity of the RNA and DNA were determined by minigel electrophoresis through a neutral 1% agarose gel. Extracted DNA and RNA were stored at 4°C and –80°C, respectively, until the time of experiments.

Probe Preparation for Chromogenic In situ Hybridization. We used the July 2003 assembly of the University of California at Santa Cruz genomic database to identify bacterial artificial chromosomes (BAC) containing the full length of VEGF (19). The probe for chromogenic in situ hybridization (CISH) was prepared from a single BAC containing VEGF (RP11-710L16). All BACs were obtained from BACPAC resources, Children's Hospital at the Oakland Research Institute and was grown according to the supplier's recommendation. DNA was isolated using Qiagen plasmid mini kit. The chromosomal location of the selected BAC was confirmed with fluorescence in situ hybridization on a normal metaphase spread (data not shown).

CISH probes were prepared from the BAC DNA using digoxigenin 12-dUTP (Roche, Indianapolis, IN) and a nick translation kit (Invitrogen). The amount of nick translation enzyme and incubation time of the reaction was adjusted to obtain an end product of 100 to 300 bp. Labeled probes were purified using Qiagen nucleotide removal kit. For each CISH experiment, 200 to 400 ng of the labeled probe was ethanol precipitated then resuspended in 10 to 20 µL of hybridization buffer (70% formamide, 2x SCC, and 10% dextran sulfate, pH 7). Probes were immediately used or stored at –20°C.

CISH Hybridization and Detection. We used the CISH polymer kit (Zymed, San Francisco, CA) for detection of the hybridized signal according to the manufacturer's protocol. Briefly, deparafinized sections were pretreated by incubation in a spotlight target retrieval solution (Zymed) at 100°C for 15minutes, followed by incubation for 1 to 15 minutes with enzyme pretreatment solution (Zymed). Slides and probes were simultaneously denatured at 97°C for 10 minutes. Hybridization was carried out at 37°C for 18 to 24 hours.

CISH signals were assessed with a Zeiss universal microscope using a 63x high dry objective. For most samples, the number of signals in 200 well-isolated nuclei were counted. The number of signals in seven different controls were used as comparison for purposes of statistical analysis. Polysomy of 6p21 was considered if the number of signal counted was more than 3 SDs generated from the seven normal control samples as previously published (20).

Comparative Genomic Hybridization. Comparative genomic hybridization analysis (CGH) was done according to published protocols (21) using the CGH nick translation labeling kit from Vysis (Downers Grove, IL). Normal reference DNA was labeled with Texas Red-5-dUTP (Applied Biosystems, Foster City, CA) and tumor DNA with fluorescein-12-dUTP (Applied Biosystems). Hybridization of normal female DNA against normal male DNA was used as a negative control. CGH images were acquired with a cooled charged-coupled device camera attached to a Zeiss Axioplan 2 epifluorescence microscope and analyzed using ISIS software (version 1.5.5, MetaSystems, Altlussheim, Germany). Data from at least 10 representative of each chromosome were combined to generate an average ratio profile reflecting the relative copy number changes in test DNA. For detection of areas of gain on 6p, we used both fixed thresholds of>1.2 and >3 SDs from the mean (21).

Quantitative RT-PCR Assay. TaqMan 5' nuclease quantitative (real time) PCR assays were carried out using predeveloped assays from Applied Biosystems according to the manufacturer's protocol with minimal modification to use the iCycler real-time PCR system (Bio-Rad, Hercules, CA). The reactions were carried out in triplicate for each sample in 25 µL with a final dilution of 1x each of Taqman PCR universal master mix (without amperase) and 1x of the predeveloped probes. In addition to VEGF, two endogenous controls, ACTB and GUS, were tested in separate reactions. The PCR reaction settings were 95°C for 3 minutes, then 40 to 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. The relative expression levels were assessed by the comparative CT method and the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by the formula 2–{Delta}{Delta}CT. The efficiency of target and reference primer-probe amplification was tested to be approximately equal in a serial dilution from the same sample.

Detection of VEGF Isoforms by RT-PCR. We used the following oligonucleotide primers, forward 5'-ATGAACTTTCTGCTGTCTTG-3' and reverse 5'-TCACCGCCTCGGCTTGTCACAT-3' in exons 1 and 8 of the VEGF gene, respectively. PCR amplification using these primers yielded products of 688,656, 584, 524, and 452 bp, which corresponded with VEGF isoforms 206, 189, 165, 145, and 121, respectively. PCR was conducted using MyCycler thermocycler (Bio-Rad). The reactions were carried out in 50 µL with a final dilution of 1x ofHotStart Taq Master Mix (Qiagen). The PCR parameters included an initial step of Taq activation at 95°C for 3 minutes followed by 40 cycles, denaturation at 95°C for 30 seconds, primer annealing at 60°C for 1 minute, and extension at 72°C for 30 seconds.

Immunostaining. Immunostaining and tissue microarray preparation were carried out at the histology core facility, Department of Pathology, Ohio State University. We used a mouse anti-human monoclonal antibody for VEGF (cloneVG1, Zymed) at a dilution of 1:200. A mouse immunoglobulin G was used as negative control. Antigen retrieval was done by heat pretreatment in a citrate buffer (pH 6) using a commercial steamer for 10 minutes. The intensity of immunostaining was scored (0-3) by two investigators (M.H. Abdel-Rahman and E.Craig) independently. Scores of 0, 1+, 2+, and 3+ were assigned for negative, mild, moderate, and strong staining of the tumor tissue cores, respectively (Fig. 1). Staining limited only to the periphery of the tissue cores were excluded from our scoring assessment. In addition, tumors with intense melanin pigmentation were excluded from analysis.



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  		Fig. 1 VEGF expression by immunohistochemistry in uveal melanoma. A, Immunostaining of tissue microarrays prepared from uveal melanomas. Three tissue microarray blocks were prepared containing representative tissues from 84 different tumors. B, C, D, and E, scoring system (0-3, respectively) of tissue cores used to grade the intensity of immunostaining. Tumors M017 (B), M107 (C), M011 (D), and M133 (E). F, immunostaining of a tissue section from tumor M011 showing moderate immunostaining of both retina and tumor. G, immunostaining of a tissue section from tumor M017 showing negative staining of the tumor but weak staining of the retina and choroid.

 
For the samples studied, we found that three different fixation and processing protocols had been utilized historically. Samples from 1979 to 1982 and from 2004 onward (28 samples) were fixed in neutral buffered formalin and processed routinely in ethanol. Samples processed in the period from 1983 to 1994 (46 samples) were fixed in formaldehyde-glutaraldehyde mixture (22) and processed in tetrahydrofuran (23). Samples collected from 1994 to 2000 (10 samples), were fixed in the formaldehyde-glutaraldehyde mixture and processed routinely in ethanol. To insure the validity of our scoring in comparison with that in the literature, we studied the variation between immunostaining intensity and the sample collection year. We identified significant differences in immunostaining with the highest frequency, 32 (69.5%) of 46, in samples from 1983 to 1994, compared with 9 (32%) of 28 and 1 (11%) of 9 in samples collected from 1979 to 1982 (P = 0.0023) and from 1996 to 2000 (P = 0.0011), respectively. These results suggest that processing samples in tetrahydrofuran enhances VEGF immunostaining. Therefore, to ensure uniformity and subjectivity of the scoring only samples fixed in neutral buffered formalin and processed routinely in ethanol (total, 28) were used for comparison of VEGF protein expression with that in the literature.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF Protein Expression by Immunohistochemistry in Ocular Melanomas. Of the 28 routinely processed ocular melanomas, we detected moderate (2+) to strong (3+) immunostaining for VEGF in 9 tumors (32%, Fig. 1). Immunostaining intensity and frequency were both much higher in samples processed in tetrahydrofuran (69.5%). Immunostaining was also carried out on whole sections from 11 different tumors including 7 tumors represented in the tissue microarray. Uniform staining of the tumor and adjacent retina was detected in all tumors with moderate to strong staining of the representative tissue cores (Fig. 1). For tumor cores scored as 1+, the immunostaining was seen in a small percentage of cells only, with weak staining of the retina. Finally, for tumors with no VEGF expression, scored as 0, immunostaining was not seen in the tumor but weak staining was seen in the adjacent retina (Fig. 1).



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  		Fig. 2 Summary of copy number change in 6p21 region detected by CGH and CISH. A, CGH profile of chromosome 6 in samples with gain in 6p. Left to right, M002, M006, M007, M017, and M4035. B, CISH using VEGF probe of sample M017. Three discrete signals could be seen in most of the cells. Inset, chromosome 6 CGH profile. C, CISH using VEGF probe of sample M4033. Only two copies of VEGF could be seen in most of the cells. Inset, chromosome 6 CGH profile.

 
VEGF mRNA Expression. Expression of VEGF mRNA was studied by quantitative RT-PCR in 15 tumors and a non–tumor blind painful eye (M028) that was included as a positive control. We used primers and probes in exons 1 and 2 of VEGF, which detectexpression of all splice variants. We operationally used the average expression level of the tumors with negative VEGF protein expression by immunohistochemistry as our "control" or baseline expression level because it is technically impossible to measure RNA expression from the nonneoplastic equivalent of ocular melanomas. We detected 7.5-fold overexpression of VEGF in 1 of 15 tumors (M011) and a 5.5-fold overexpression in the nontumor control (M028). In 9 of the 16 samples with mRNA expression data, we were also able to estimate VEGF protein expression by immunohistochemistry. Moderate immunostaining for VEGF was detected in samples M011 and M028 (Fig. 2). In general, VEGF transcript expression levels and VEGF protein expression in routinely fixed and processed samples were concordant (Fig. 3).



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  		Fig. 3 Expression of VEGF mRNA in uveal melanoma. A, quantitative real-time RT-PCR of 15 different uveal melanomas and one blind, painful eye (M028). The expression level of VEGF was assessed in triplicate. VEGF expression was normalized relative to GUSB expression in the same samples. The presented VEGF fold change was quantified relative to the average expression level in tumors negative to VEGF protein expression by immunostaining (see text for details). Immunostaining was carried out for nine samples (stars). Filled stars, samples with positive immunostaining of VEGF (M011 and M028). B, different splice variants of the VEGF could be detected in different tumors. Overall, VEGF mRNA expression could be detected in most of the tumors. In sample M011, the overexpression is limited to splice variant VEGF165, whereas in samples M05 and M017, other splice variants (VEGF189, VEGF165, and VEGF121) are also expressed and VEGF121 is relatively overexpressed. Expression of ß actin (ACTB) was used as a control for efficiency of cDNA synthesis.

 
VEGF mRNA splice variants were assessed by RT-PCR using primers in exons 1 and 8 of the gene. We detected expression of splice variants VEGF189, VEGF165, and VEGF121 in all the samples expressing the gene. In samples M005 and M017, the expression of VEGF121 was prominent. In samples M011, M06, and M030, the expression of VEGF165 was the most prominent. In sum, there was no clear correlation between expression of isoforms and VEGF protein expression.

Assessment of VEGF/6p21 Genomic Copy Number in Ocular Melanoma. A total of 37 distinct ocular melanomas could be assessed for 6p21 copy number by CISH (n = 26), CGH (n = 14), or both (n = 3). Of these 37, 10 (27%), 7 by CISH alone,2 by CGH alone, and 1 by both techniques were shown tohave overrepresentation of that region. Among the 8 detected by CISH, polysomy ranged from 3 to 9 copies per tumor cell (Table 1). Three of these 8 tumors showed a significant number ofcells with more than 5 copies of VEGF. In addition to the 3 tumors with gain in the 6p21 region detected by CGH, 2 other tumors (M002 and M006) showed gain of 6p that did not involve the 6p21 region (Fig. 2). However, in one of these tumors (M006), genetic alteration of the 6p21 region was detected by genotyping, suggesting a genetic alteration below the threshold of detection of CGH (data not shown). The correlation between CISH and CGH data is illustrated for samples M017 and M4033 (Fig. 2). Of note, there was no statistically significant difference in VEGF expression between tumors with (5/26) and without (2/10) overrepresentation of 6p21 (P = 0.82).


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  		Table 1 VEGF expression in tumors with overrepresentation of 6p21 region

 

   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
REFERENCES
 
We found overrepresentation of the VEGF/6p21 genomic region in 9 (25%) of 36 uveal melanomas. This is a figure that is slightly lower than the 31% to 57% frequency of reported gains in the 6p region (12–14, 16). Interestingly, our CGH data suggest that the smallest region of gain on 6p is 6p25-pter, which is telomeric to and excludes the chromosomal location in which VEGF resides (Fig. 2). Although we detected polysomy of the VEGF genomic region by CISH in 8 (32%) of 25 tumors, only 3 showed significant numbers of tumor cells with 5 copies or more of VEGF/cell. Overexpression of VEGF was detected in 2 of 3 tumors with ≥5 copies of VEGF. However, in 5 tumors with no alteration of the VEGF copy number, overexpression of VEGF was also detected. Therefore, our observations show that overexpression of VEGF in ocular melanomas can occur without alteration in the VEGF copy number. These data suggest that VEGF overexpression due to mechanisms other than structural amplification is probably significant in the pathogenesis of uveal melanomas. In this regard, VEGF has been shown to be elevated in the aqueous or vitreous of eyes harboring uveal melanomas independent of its expression in the corresponding tumors (24). Taken together with this report, our current observations lead us to postulate that although VEGF may play a prominent role in ocular melanomas, the actual type of cells producing VEGF may be quite diverse. It would seem that overexpression of VEGF in a minority of ocular melanomas is indeed due to overrepresentation of VEGF gene copy number in the tumor itself (this study). However, epigenetic mechanisms of VEGF overexpression in ocular melanomas would also be consistent with our observations. Furthermore, we also postulate that VEGF production by normal tissue or cells in the microenvironment of the tumor would be germane to ocular melanoma behavior, irrespective of VEGF expression in the tumor per se. Finally, our observation of overrepresentation of the 6p25-pter region, telomeric of the VEGF gene, is noteworthy. Our data could suggest that a gene or genes residing telomeric of VEGF, when over represented may play a role in the pathogenesis of ocular melanoma. This should be further investigated.

The previously reported frequency of VEGF expression in uveal melanoma is highly variable (9–11, 25). The variation in the fixation and processing techniques may be a potential explanation that our study has revealed. The samples included in this study were collected from the same institution over a period of 20 years. During those years, fixation and processing have changed twice, which was reflected in both the immunostaining results and the efficiency of in situ hybridization. In our study, VEGF expression in routinely fixed tissues, 9 (32%) of 28, was similar to frequencies previously published (11). The high frequency, 32 (69.5%) of 46, of immunostaining observed in samples processed in tetrahydrofuran may suggest that this type of processing enhances VEGF protein detection by immunohistochemistry.

Another important factor that may explain the variation in the reported frequencies of VEGF expression may be the specificity of the antibody to different VEGF splice variants. Our RT-PCR results indicate that different VEGF splice variants are expressed in uveal melanomas. In our study, we used a monoclonal antibody, clone VG1, which reacts with the 121, 165, and 189 isoforms of VEGF protein in routinely processed material (26). Most of the other published studies used polyclonal antibodies with unknown specificity to VEGF splice variants (9–11).

In conclusion, VEGF overexpression could be detected in about 32% of routinely fixed and processed uveal melanomas. Of note, VEGF expression can be independent of the copy number of VEGF. Therefore, VEGF overexpression due to pathways other than structural amplification is probably significant in the pathogenesis of uveal melanomas, and its precise mechanism must be sought.


   FOOTNOTES
 
Grant support: Patti Blow's research fund in Ophthalmology and National Cancer Institute grant P30CA16058 to the Ohio State University Comprehensive Cancer Center, and Department of Internal Medicine Selective Investment fund (M.H. Abdel-Rahman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/14/04; revised 9/14/04; accepted 9/21/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 

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