January 2010
Volume 51, Issue 1
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Anatomy and Pathology/Oncology  |   January 2010
Proteomics of Uveal Melanomas Suggests HSP-27 as a Possible Surrogate Marker of Chromosome 3 Loss
Author Affiliations & Notes
  • Sarah E. Coupland
    From the Department of Pathology, School of Cancer Studies, University of Liverpool, Liverpool, United Kingdom;
  • Henrik Vorum
    the Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark;
    the Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark; and
  • Nakul Mandal
    the Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark; and
  • Helen Kalirai
    From the Department of Pathology, School of Cancer Studies, University of Liverpool, Liverpool, United Kingdom;
  • Bent Honoré
    the Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark; and
  • Steen Fiil Urbak
    the Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark;
  • Sarah L. Lake
    From the Department of Pathology, School of Cancer Studies, University of Liverpool, Liverpool, United Kingdom;
  • Justyna Dopierala
    From the Department of Pathology, School of Cancer Studies, University of Liverpool, Liverpool, United Kingdom;
  • Bertil Damato
    the Ocular Oncology Service, St. Paul's Eye Clinic, Royal Liverpool University Hospital, Liverpool, United Kingdom.
  • Corresponding author: Sarah E. Coupland, Department of Pathology, University of Liverpool, Liverpool, UK; s.e.coupland@liverpool.ac.uk
  • Footnotes
    2  Contributed equally to the work and therefore should be considered equivalent authors.
  • Footnotes
    5  Present affiliation: Department of Ophthalmology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark.
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 12-20. doi:10.1167/iovs.09-3913
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      Sarah E. Coupland, Henrik Vorum, Nakul Mandal, Helen Kalirai, Bent Honoré, Steen Fiil Urbak, Sarah L. Lake, Justyna Dopierala, Bertil Damato; Proteomics of Uveal Melanomas Suggests HSP-27 as a Possible Surrogate Marker of Chromosome 3 Loss. Invest. Ophthalmol. Vis. Sci. 2010;51(1):12-20. doi: 10.1167/iovs.09-3913.

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Abstract

Purpose.: To compare the proteomic profiles of primary uveal melanomas, with and without loss of chromosome 3.

Methods.: Frozen specimens from three uveal melanomas with disomy 3 and from four tumors with monosomy 3, according to fluorescence in situ hybridization (FISH) analysis, were subjected to high-resolution, two-dimensional (2-D) gel electrophoresis. The protein expression profiles of the two uveal melanoma cytogenetic groups were compared: Proteins that differed significantly were excised and analyzed by tandem mass spectrometry. Differentially expressed proteins were further analyzed with Western blot analysis. An independent cohort of 41 formalin-fixed, paraffin-embedded (FFPE) uveal melanomas, whose chromosome 3 status had been determined by multiplex ligation-dependent probe amplification (MLPA), was examined for the appropriate antigens by immunohistochemistry.

Results.: Four protein spots were 1.5-fold (Student's t-test, P < 0.05) differentially expressed in the two uveal melanoma types: two spots were overexpressed in the disomy 3 group compared with the monosomy 3 group, whereas two spots were underexpressed. Identification of the four spots yielded nine proteins. Western blot analysis confirmed the results for heat shock protein (HSP)-27, vimentin, and pyruvate dehydrogenase β (PDHB), with a statistical significance for the first two proteins. HSP-27 was significantly downregulated, whereas vimentin was upregulated in the monosomy 3 tumors (Student's t-test, P = 0.003 and P = 0.005, respectively). Immunohistochemistry confirmed low-to-negative HSP-27 protein expression in monosomy 3 uveal melanomas (Student's t-test; P = 0.011).

Conclusions.: Low-to-negative HSP-27 protein expression in uveal melanoma correlates strongly with monosomy 3. Further validation is necessary to determine whether immunohistochemical assessment of HSP-27 expression correlates with metastatic mortality.

Uveal melanoma causes metastatic disease in approximately 50% of all patients, despite successful treatment of the primary intraocular tumor. 1 Metastatic disease usually involves the liver and is invariably fatal, typically within a year of the onset of symptoms. 2,3 Uveal melanomas can essentially be subtyped into low- and high-grade metastatic risk groups, according to their clinical, histologic and genotypic features. 26 Knowledge of a particular tumor's characteristics allows for a more accurate prediction of metastasis in the individual patient and aids treatment and management decisions. 2,3  
Although a large amount of information is available with regard to the molecular biological differences of low- and high-grade uveal melanomas, little is known about the corresponding protein complement of the genome (proteomics) in these melanoma subgroups. To date, proteomic analysis has been conducted on uveal melanoma cell lines, 79 serum, 10 and aqueous humor 11 of patients with uveal melanoma. These investigations revealed overexpression and underexpression of some proteins; however, no correlation was made with the cytogenetic status of the tumor cells. 
The purpose of our study was, therefore, to determine whether the proteomic profiles of monosomy 3 uveal melanomas differed significantly from those of disomy 3 tumors and, if so, whether such differences identified possible biomarkers of the high-grade uveal melanomas. 
Methods
The study was approved by the local committee of the National Research Ethics Service and informed consent was obtained from all patients. The research adhered to the tenets of the Declaration of Helsinki. 
Proteomic Analysis
Seven uveal melanomas were selected for proteomic analysis. These frozen tumor samples had been taken from uveal melanomas treated by local resection or enucleation in 2005 and had been selected on the basis of cytogenetic analysis for chromosome 3 routinely performed by fluorescence in situ hybridization (FISH) at this time. 2 The histomorphologic features of these seven uveal melanomas were noted after routine fixation, embedding in paraffin, and staining of sections, using hematoxylin and eosin (H&E) and periodic acid Schiff (PAS). 
Protein Extraction.
Frozen uveal melanoma tissue was homogenized and dissolved in lysis buffer containing 9 M urea, 2% (vol/vol) Triton X-100, 2% (vol/vol) immobilized pH gradient (IPG) buffer (pH 3-10NL), and 2% (wt/vol) dithiothreitol (DTT). The protein samples were stored at −80°C. The total protein content in each melanoma sample was assayed (Non-interfering Protein Assay; Calbiochem-Merck Chemicals, Beeston, UK). 12  
Two-Dimensional Gel Electrophoresis.
One-dimensional isoelectric focusing (IEF) was performed by using nonlinear pH 3-10NL IPG strips (Amersham Pharmacia Biotech, Uppsala, Sweden). The IPG strip was rehydrated for 20 hours at room temperature in 200 μL lysis buffer with approximately 100 μg protein, and 150 μL rehydration buffer (8 M urea, 2% [wt/vol] CHAPS, 0.3% [wt/vol] DTT, and 2% [vol/vol] IPG buffer), using a dry strip tray (Immobiline DryStrip Reswelling Tray; Amersham Pharmacia Biotech). IEF was performed on an electrophoresis unit (Multiphor II; PerkinElmer Windsor, UK) at 500 V for 0.01 hour, 500 V for 5 hours, 3500 V for 5 hours, and 3500 V for 9.5 hours in a gradient mode at 17°C (MultiTemp III Thermostatic Circulator; GE Healthcare Life Sciences, Buckinghamshire, UK). Before two-dimensional SDS-PAGE, the IPG strip was equilibrated twice: first for 10 minutes under gentle agitation in 20 mL of equilibration solution containing 0.6% (wt/vol) Tris-HCl (pH 6.8), 6 M urea, 30% (vol/vol) glycerol, 1% (wt/vol) SDS, and 0.05% (wt/vol) DTT; and second in 4.5% (wt/vol) iodoacetamide and bromophenol blue. For the second dimension, the equilibrated IPG strip was transferred to a polyacrylamide gel. Electrophoresis was then performed vertically at a maximum voltage of 50 V, 5 mA for approximately 20 hours on equipment designed and previously described by Vorum et al. 13  
Silver Staining.
Gels were visualized by silver staining optimized for high-sensitivity protein identification by mass spectrometry. 14 Briefly, individual gels were fixed in 50% (vol/vol) ethanol, 12% (vol/vol) acetic acid, and 0.0185% (vol/vol) formaldehyde overnight. After the gels were washed three times for 20 minutes in 35% (vol/vol) ethanol and pretreated for 1 minute in 0.02% (wt/vol) Na2S2O3;5H2O, they were rinsed in water and stained in 0.2% (wt/vol) AgNO3 and 0.028% (vol/vol) formaldehyde for 20 minutes. Further rinsing with water was performed before development in 6% (wt/vol) Na2CO3 and 0.0185% (vol/vol) formaldehyde, 0.0004% (wt/vol) Na2S2O3;5H2O, for approximately 3 minutes. Finally, development was arrested in a fixative solution of 40% (vol/vol) ethanol, 12% (vol/vol) acetic acid. 
Image Analysis.
Silver-stained gels were scanned with a densitometer (GS-710 Calibrated Imaging Densitometer; Bio-Rad, Hemel Hempstead, UK). The peptide spots were analyzed by software (PDQuest software; Bio-Rad) that designated a volume to each spot proportional to the amount of protein. All well-separated and clearly focused spots that were 1.5-fold (Student's t-test P < 0.05) differentially expressed between the disomy 3 and monosomy 3 uveal melanomas were selected for identification by liquid chromatography–tandem mass spectrometry. 12  
Protein Identification.
The proteins were excised from the gels and subjected to in-gel digestion. The peptide samples were analyzed by LC-MS/MS essentially as described. 12 In short, digested peptides were separated on an inert nano liquid chromatography system (LC Packings, San Francisco, CA) connected to a mass spectrometer (Q-Tof Premier; Waters, Milford, MA), spectra were obtained (MassLynx 4 SP4; Waters), and raw data were processed (ProteinLynx GlobalServer 2.1; Waters). The processed data were used to search the total part of the Swiss-Prot database (release 52.4, 265,950 sequences; http://www.expasy.org; provided in the public domain by Swiss Institute of Bioinformatics, Geneva, Switzerland) using the online version of the Mascot MS/MS Ion Search facility (http://www.matrixscience.com/ available commercially from Matrix Science, Ltd., London, UK). The search was performed with doubly and triply charged ions with two missed cleavages, a peptide tolerance of 50 ppm, one variable modification, carbamidomethyl-C, and an MS/MS tolerance of 0.05 Da. Contaminating peptides, such as keratins and trypsin, cross-contaminating peptides from previous samples, and peptides originating from different sources such as BSA, were disregarded. At least one bold red peptide was required in the search. The scores of the protein hits were recorded and are reported herein. Individual peptide ion scores higher than 36 indicate identity or extensive homology, giving a less than 5% probability that the observed match was a random event. 
Western Blot Analysis
Samples were loaded and run on 10% to 20% gradient Tris-Glycine polyacrylamide gels (Novex; Invitrogen Corp.; Paisley, Scotland, UK). After transfer by electroelution to nitrocellulose membranes (Hybond-C Extra; Amersham, UK), the blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20 (PBS-T; pH 7.5) for 1 hour and incubated overnight at 4°C with the appropriate primary antibodies (Table 1). The labeling was visualized with horseradish peroxidase (HRP)–conjugated secondary antibodies (P0217, P0449, or P0260; DakoCytomation, Glostrup, Denmark, and ab16349-250; Abcam, Cambridge, UK) by enhanced chemiluminescence (GE Healthcare). In an effort to conserve tissue samples, some blots were reprobed after treatment with Western blot stripping buffer according to the manufacturer's protocol (Restore; Pierce Biotechnology Inc., Rockford, IL). 
Table 1.
 
Commercial Antibodies Used in Western Blot Analysis
Table 1.
 
Commercial Antibodies Used in Western Blot Analysis
Antibody Name Clone/Peptide Species Manufacturer Antibody Concentration
Heat shock protein 27 Polyclonal; C terminus Goat Santa Cruz Biotechnology, Santa Cruz, CA 1:100
Vimentin Polyclonal; C terminus Rabbit Santa Cruz Biotechnology 1:100
Pyruvate dehydrogenase β Monoclonal; partial recombinant Mouse Abnova Corp., Taipei, Taiwan 1:100
β-Actin Polyclonal; aa 151–320 Chicken Abcam, Cambridge, UK 1:500
Glyceraldehyde-3-phosphate dehydrogenase Polyclonal; internal Goat Santa Cruz Biotechnology Inc. *
Glyceraldehyde-3-phosphate dehydrogenase Polyclonal; C terminus Rabbit GenWay Biotech Inc., San Diego, CA *
Fumarate hydratase Polyclonal; recombinant Rabbit Proteintech Group Inc., Chicago, IL *
α-Tubulin Polyclonal; N terminus Goat Santa Cruz Biotechnology *
Tropomyosin Polyclonal; aa 1-284 Rabbit Santa Cruz Biotechnology *
Lysozyme C Polyclonal; Internal Goat Santa Cruz Biotechnology *
F-actin capping protein α subunit (CAPZA 1) Polyclonal; aa 1-286 Chicken Abcam *
Immunohistochemistry
Antigens identified as being significantly differentially expressed between monosomy 3 and disomy 3 tumors in the proteomic studies were further investigated by immunohistochemistry applied to an independent cohort of 41 uveal melanomas. These 41 tumors were selected on the basis of their chromosome 3 status, as determined for prognostication purposes by MLPA 4 in a fresh biopsy taken directly after enucleation or tumor excision between 2006 and 2008. 
After conventional staining of these 41 uveal melanomas with H&E and PAS, immunohistochemistry was performed on 4-μm sections made from the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Sections were dewaxed, rehydrated, and endogenous peroxidase blocked with H2O2 in methanol. To detect HSP-27, we also subjected the slides to heat-induced epitope retrieval (pH 7.0) before incubation with the appropriate antibody. After they were rinsed in running water, slides were loaded onto an autostainer (DakoCytomation) and washed with phosphate-buffered saline (PBS). The sections were incubated with primary antibody for 40 minutes at room temperature (RT): mouse anti-human HSP-27 antibody, diluted 1:100 (Novocastra, Newcastle, UK), and mouse anti-human vimentin antibody, 1 μg/mL (DakoCytomation). Bound antibody was detected with an HRP system (Advance; DakoCytomation) according to the manufacturer's instructions. Peroxidase was visualized with an AEC substrate kit (Vector Laboratories Ltd, Peterborough, UK), and the sections were counterstained with Mayer's hematoxylin and mounted in aqueous medium (Aquatex; Merck Chemicals, Ltd., Nottingham, UK). Negative control experiments were performed by replacing the primary antibody with mouse isotype control antibody. Normal human skin and tonsil were used as positive control tissues for HSP-27 and vimentin antibodies, respectively. 
The percentages of melanoma cells staining for HSP-27 and vimentin were evaluated by two independent observers who were unaware of the chromosome 3 status. These percentages were scored as follows: 0% tumor cells positive (score 0), 1% to 24% tumor cells positive (score 1), 25% to 49% tumor cells positive (score 2), 50% to 74% tumor cells positive (score 3), and 75% to 100% tumor cells positive (score 4). The intensity of cellular staining was also scored as none (0), weak (1), moderate (2), or strong (3). A combined score was thus obtained by multiplying the two scores, as previously described in other tumors, 15 giving a possible maximum score of 12. 
Multiplex Ligation-Dependent Probe Amplification (MLPA)
The genomic abnormalities of the independent cohort of 41 uveal melanomas were determined with MLPA, as described previously. 4,16 MLPA replaced FISH as the diagnostic test used to identify chromosomal aberrations at the Royal Liverpool Hospital Ocular Oncology Centre in 2007, since, as detailed in our recent report, 4 it accurately detects monosomy 3 with a greater sensitivity than FISH. 
Statistical Analysis
Differences in the protein expression levels for HSP-27 and vimentin were examined with a Student's t-test between the uveal melanoma subgroups (i.e., monosomy 3 versus disomy 3). This test was also used for any correlations with histomorphologic features (e.g., cell type and presence or absence of closed loops). Correlations between the protein expression levels for HSP-27 or vimentin and age at primary diagnosis, largest basal diameter (LBD), tumor height, and mitotic frequency were determined with the bivariate Spearman's correlation (all statistical analysis by SPSS, ver.11.0; SPSS Science, Chicago, IL). 
Results
Differential Protein Expression According to the Presence or Absence of Monosomy 3
The proteomic profiles of three disomy 3 uveal melanomas were compared with those of four monosomy 3 tumors. The clinical, histopathologic, and cytogenetic features of these tumors are summarized in Table 2. Two protein spots were overexpressed in the disomy 3 melanomas compared with the monosomy 3 tumors, whereas two spots were underexpressed (Fig. 1). In four spots, tandem mass spectrometry identified nine proteins, as indicated in Table 3. Western blot analysis of the protein extracts showed significant differences for HSP-27 and vimentin but not pyruvate dehydrogenase β (PDHB; Fig. 2) between the uveal melanoma groups. Compared to disomy 3 uveal melanomas, monosomy 3 tumors showed downregulation of HSP-27 and upregulation of vimentin (Fig. 2; Table 4). 
Table 2.
 
Clinicopathologic Data of Patients from the Proteomic Analysis
Table 2.
 
Clinicopathologic Data of Patients from the Proteomic Analysis
Sample ID Sex Age (y) Cell Type LBD (mm) Height (mm) Mitosis (no./40 HPF) Closed Loops Chromosome 3 Status*
IOM001 F 56 E 15.8 13.7 1 N D
IOM002 F 30 E 15.9 10.1 9 N D
IOM006 F 81 E 11.4 7.6 2 N D
IOM003 M 70 S 19.2 9.2 7 Y M
IOM004 F 42 E 19.2 12.7 28 N M
IOM005 F 78 E 20.0 9.7 2 Y M
IOM007 M 49 E 17.5 7.5 6 N M
Figure 1.
 
Two-dimensional polyacrylamide gel analysis. Histograms show the densitometry measurements of the significantly up- or downregulated spots. Blue: disomy 3; red: monosomy 3.
Figure 1.
 
Two-dimensional polyacrylamide gel analysis. Histograms show the densitometry measurements of the significantly up- or downregulated spots. Blue: disomy 3; red: monosomy 3.
Table 3.
 
Mass Spectrometric Analysis of Differentially Expressed Protein Spots
Table 3.
 
Mass Spectrometric Analysis of Differentially Expressed Protein Spots
Spot M3/D3 Ratio ID MW Score
0306 3.08 Tropomyosin α-4 chain TPM4_HUMAN 28.5 551
Vimentin VIME_HUMAN 53.6 343
Lysozyme C LYSC_HUMAN 16.5 75
3410 0.53 Tubulin TBAK_HUMAN 50.1 526
Pyruvate dehydrogenase El component β subunit (mitochondrial) ODPB_HUMAN 39.2 126
F-capping protein subunit α-1 CAZA1_HUMAN 32.9 36
5213 0.47 Heat-shock protein β-1 (HSP 27) HSPB1_HUMAN 22.8 572
8006 2.17 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) G3P_HUMAN 36.0 164
Fumarate hydratase (mitochondrial) FUMH_HUMAN 54.6 41
Figure 2.
 
Western blot analysis of uveal melanoma tissue: three tumors with disomy 3 and four tumors with monosomy 3. (A) Blots were incubated with commercial antibodies against the appropriate antigens. β-Actin was used as a loading control. (B) Mean densitometry (optical density × area) of the antibody–antigen reaction shown on the blots. Student's two-tailed t-test: *Only the two strongest visible reactions in the PDHB disomy 3 were used in the calculations.
Figure 2.
 
Western blot analysis of uveal melanoma tissue: three tumors with disomy 3 and four tumors with monosomy 3. (A) Blots were incubated with commercial antibodies against the appropriate antigens. β-Actin was used as a loading control. (B) Mean densitometry (optical density × area) of the antibody–antigen reaction shown on the blots. Student's two-tailed t-test: *Only the two strongest visible reactions in the PDHB disomy 3 were used in the calculations.
Table 4.
 
Western Blot Densitometry
Table 4.
 
Western Blot Densitometry
Antibody Disomy 3 Mean Volume (ODxArea) Monosomy 3 Mean Volume (ODxArea) P (Student's t-test) Factor Up-/Down- regulation Monosomy 3
Vimentin 5.82 12.20 0.005 X2.1 UP
HSP-27 10.51 6.67 0.003 X1.6 DOWN
PDHB 3.31* 2.53 0.097 X1.3 DOWN
Expression of HSP-27 and Vimentin Protein in Sections of Uveal Melanoma
HSP-27 and vimentin protein expression were determined by immunohistochemistry in FFPE sections of an independent cohort of 41 uveal melanomas treated between 2006 and 2008, either by local resection or enucleation. Twenty-one of these tumors had been classified as disomy 3 and 20 as monosomy 3 after MLPA analysis of the corresponding diagnostic frozen tumor specimen. 
HSP-27 Immunoreactivity.
The intrinsic positive control for HSP-27 in the enucleated eyes was the pigmented and nonpigmented ciliary body epithelium, which demonstrated cytoplasmic staining, whereas the intrinsic negative controls included macrophages and lymphocytes within the tumors. HSP-27 staining was also cytoplasmic in the uveal melanoma cells. The Mayer's hematoxylin counterstain facilitated assessment of the cellular morphology, and, therefore, determination of immunopositivity in either reactive or tumor cells. HSP-27 positivity ranged from 0% to 100% (mean, 64.0%) with tumor cells displaying various degrees of intensity between specimens (Fig. 3A–D; Table 5). HSP-27 staining also showed some intratumoral variation—for example, tending to be stronger in melanoma cells surrounding blood vessels and showing more heterogeneity in areas of epithelioid cells (Fig. 3). 
Figure 3.
 
HSP-27 immunoreactivity in FFPE uveal melanomas. (A) The tumor cells, which are of the epithelioid type, demonstrated a strong and homogenous positivity for HSP-27. (BD) Heterogeneous immunoreactivity for HSP-27 in uveal melanomas, depending on cell type, admixed reactive cells, and distance from blood vessels; (B) mixed-cell uveal melanoma; (C) epithelioid cell type uveal melanoma with admixed melanomacrophages; and (D) spindle cell type uveal melanoma. Magnification: (A) ×40 objective; (B, D) ×20 objective; (B inset; C) ×60 objective.
Figure 3.
 
HSP-27 immunoreactivity in FFPE uveal melanomas. (A) The tumor cells, which are of the epithelioid type, demonstrated a strong and homogenous positivity for HSP-27. (BD) Heterogeneous immunoreactivity for HSP-27 in uveal melanomas, depending on cell type, admixed reactive cells, and distance from blood vessels; (B) mixed-cell uveal melanoma; (C) epithelioid cell type uveal melanoma with admixed melanomacrophages; and (D) spindle cell type uveal melanoma. Magnification: (A) ×40 objective; (B, D) ×20 objective; (B inset; C) ×60 objective.
Table 5.
 
Clinicopathologic Parameters and HSP-27 and Vimentin Protein Expression Data
Table 5.
 
Clinicopathologic Parameters and HSP-27 and Vimentin Protein Expression Data
Sample ID Age at Primary Diagnosis (y) Cell Type LBD (mm) Height (mm) Mitotic Frequency (No./40 HPF) Closed Loops Chromosome 3 Status (MLPA) HSP-27 Vimentin
% Tumor Cells Stained Intensity of Tumor Cells Stained Staining Score % Tumor Cells Stained Intensity of Tumor Cells Stained Staining Score
1 44 S 17.3 11.0 2 N D 20 Moderate 2 55 Weak 3
2 72 S 12.2 11.1 14 N D 15 Moderate 2 50 Moderate 6
3 80 S 15.1 10.7 2 Y D 90 Moderate 8 40 Moderate 8
4 63 S 16.1 7.5 7 Y D 55 Moderate 6 65 Moderate 6
5 69 S 12.0 3.7 3 N D 100 Moderate 8 10 Moderate 2
6 62 E 14.4 6.4 4 Y D 90 Strong 12 90 Strong 12
7 52 S 21.2 10.8 4 Y D 40 Moderate 4 85 Strong 4
8 40 S 17.5 7.9 7 N D 60 Moderate 6 90 Weak 4
9 74 S 7.8 1.9 1 N D 100 Moderate 8 90 Weak 4
10 81 S 15.3 9.5 4 N D 50 Moderate 6 75 Weak 4
11 42 S 16.7 9.1 1 N D 65 Strong 9 80 Weak 4
12 34 S 9.6 6.9 7 N D 90 Moderate 8 95 Moderate 8
13 80 S 15.1 10.7 2 Y D 95 Strong 12 95 Moderate 8
14 53 S 18.5 13.0 2 N D 75 Moderate 8 50 Moderate 6
15 67 S 17.0 9.2 2 Y D 100 Strong 12 70 Moderate 6
16 67 S 14.9 10.5 6 N D 85 Moderate 8 100 Weak 4
17 80 S 20.6 15.7 15 Y D 90 Moderate 8 95 Weak 4
18 49 S 18.3 11.7 1 N D 95 Moderate 8 60 Moderate 6
19 60 S 14.8 3.1 4 Y D 95 Moderate 8 75 Weak 4
20 46 S 12.3 4.5 4 Y D 100 Moderate 8 100 Strong 12
21 62 S 11.1 4.0 2 N D 100 Strong 12 95 Strong 12
22 67 E 22.1 10.2 31 N M 75 Moderate 8 40 Weak 2
23 62 E 19.1 8.4 7 Y M 5 Weak 1 90 Moderate 8
24 66 E 16.4 6.6 8 Y M 10 Weak 1 85 Weak 4
25 59 E 20.0 7.5 6 Y M 10 Moderate 2 95 Moderate 8
26 63 E 17.5 10.3 13 N M 15 Moderate 2 30 Moderate 4
27 44 E 13.6 6.7 7 Y M 0 Nonee 0 85 Moderate 8
28 62 E 19.1 6.8 15 Y M 75 Moderate 8 95 Moderate 8
29 48 S 15.6 8.1 9 Y M 0 Nonee 0 95 Weak 4
30 68 E 17.3 11.7 10 N M 65 Weak 3 45 Weak 2
31 71 S 17.2 12.6 7 N M 15 Moderate 2 65 Moderate 6
32 77 S 15.2 9.7 4 N M 30 Moderate 4 80 Moderate 8
33 53 S 15.2 9.7 2 Y M 85 Weak 4 80 Moderate 8
34 61 E 16.3 7.2 11 Y M 55 Moderate 6 95 Weak 4
35 53 E 20.0 8.3 12 Y M 25 Weak 2 85 Moderate 8
36 63 S 17.3 10.3 2 N M 85 Moderate 8 90 Weak 4
37 73 E 14.8 7.7 3 Y M 95 Strong 12 90 Moderate 8
38 53 S 16.6 13.6 4 N M 85 Moderate 8 80 Weak 4
39 64 E 19.6 9.8 17 Y M 85 Moderate 8 90 Weak 4
40 62 S 17.4 7.3 2 N M 90 Moderate 8 50 Weak 3
41 50 E 14.5 7.5 14 Y M 100 Strong 12 95 Moderate 8
Vimentin Immunoreactivity.
An intrinsic positive control for vimentin in the enucleated eyes was the retina, demonstrating positivity within the Müller cells and similar support cells. Vimentin staining was cytoplasmic and ranged from 10% to 100% (mean; 76.0%) within the tumor cells. As with HSP-27 staining, vimentin protein expression showed some intratumoral variation (Fig. 4). 
Figure 4.
 
Vimentin immunoreactivity in FFPE uveal melanomas. (A) The melanoma cells, which are of the spindle type, demonstrated a strong immunoreactivity for vimentin. (B, C) Heterogenous immunoreactivity for vimentin was present in these uveal melanomas (of epithelioid and mixed cell type, respectively), depending on cell type, the presence/absence of closed loops (inset, PAS without counterstain), and the distance from blood vessels. Magnification: (A, B inset, C) ×20 objective; (B) ×40 objective.
Figure 4.
 
Vimentin immunoreactivity in FFPE uveal melanomas. (A) The melanoma cells, which are of the spindle type, demonstrated a strong immunoreactivity for vimentin. (B, C) Heterogenous immunoreactivity for vimentin was present in these uveal melanomas (of epithelioid and mixed cell type, respectively), depending on cell type, the presence/absence of closed loops (inset, PAS without counterstain), and the distance from blood vessels. Magnification: (A, B inset, C) ×20 objective; (B) ×40 objective.
Correlation of HSP-27 and Vimentin Protein Expression with Clinicopathologic and Cytogenetic Risk Factors for Disease Progression and Metastasis
Clinicopathologic information for all patients included in this study is detailed in Table 5. Briefly, these 41 patients with uveal melanoma had a mean age of 60.9 years (range, 34.0–81.0) at primary treatment. All patients had been treated by enucleation or local resection (i.e., no uveal melanoma had undergone radiotherapy). The tumors had a mean largest basal diameter of 16.2 mm (range, 7.8–22.1) and a mean height of 8.8 mm (range, 1.9–15.7). Histologic examination, as described previously, 2 showed epithelioid cells in 14 tumors and closed PAS+ loops in 21 tumors, with the mitotic rate exceeding 5 per 40 high-power fields in 20 tumors. 
Statistical analysis of the HSP-27 protein expression score (i.e., percentage of tumor cells stained multiplied by intensity scores) was significantly lower in monosomy 3 uveal melanoma when compared with disomy 3 tumors (Student's t-test; P = 0.011; Fig. 5A). This remained statistically significant even if only the percentage of tumor cells stained was tested (Student's t-test; P = 0.013; Fig. 5B). Neither the vimentin protein expression score nor the percentage of tumor cells stained showed significant differences between monosomy 3 and disomy 3 tumors (Student's t-test; P = 0.64 and P = 0.60, respectively). 
Figure 5.
 
Boxplots showing the relationship between chromosome 3 status and (A) the HSP-27 protein expression score (B) the percentage of tumor cells that stained positively for HSP-27. Probabilities show the statistical significance of the differences between monosomy 3 (M3; n = 20) and disomy 3 (D3; n = 21) uveal melanomas, as determined by Student's t-test.
Figure 5.
 
Boxplots showing the relationship between chromosome 3 status and (A) the HSP-27 protein expression score (B) the percentage of tumor cells that stained positively for HSP-27. Probabilities show the statistical significance of the differences between monosomy 3 (M3; n = 20) and disomy 3 (D3; n = 21) uveal melanomas, as determined by Student's t-test.
With respect to the histomorphologic features, statistical analysis of the HSP-27 protein expression score in uveal melanomas showed a negative correlation with mitotic frequency (Spearman's correlation; P = 0.014). The vimentin protein expression score was significantly higher in uveal melanomas with closed loops than in those without (Student's t-test; P = 0.011). In addition, a negative correlation was observed between the vimentin protein expression score and LBD (Spearman's correlation; P = 0.043). Neither HSP-27 nor vimentin immunoreactivity showed a significant association with any other clinicopathologic variable tested (i.e., age of patient at primary diagnosis, cell type, or tumor height). 
Discussion
The present study demonstrates a statistically significant association between chromosome 3 loss in uveal melanomas and the downregulation of HSP-27 (P = 0.011). This association was demonstrated with both proteomics and immunohistochemistry. To our knowledge, this correlation has not been reported previously. Our study raises the possibility of using immunohistochemical staining for HSP27 as a prognostic indicator when cytogenetic studies are not possible. 
HSPs are a class of functionally related proteins with expression that increases when cells are exposed to elevated temperatures or stress. Intracellular HSPs, including HSP-27 (located on chromosome 7), are overexpressed in a variety of cancer cells, are essential for the survival of these cell types, and have been demonstrated as being prognostically significant in some carcinomas. For example, HSP-27 overexpression is associated with a poor prognosis in gastric, prostate, and node-negative breast carcinomas. 1719 In contrast, high levels of HSP-27 expression indicate a good prognosis in non–small-cell lung carcinomas and ovarian carcinomas. 20,21  
HSPs have been reported previously in a limited number of uveal melanomas and cell lines. 7,9,22 Pardo et al. 7 described several HSPs with the exception of HSP-27, in the proteome of a single patient with uveal melanoma. Zuidervaart et al. 9 reported relative HSP-27 overexpression in a cell line derived from a uveal melanoma metastasis compared with the cell line derived from the corresponding primary uveal melanoma. High levels of HSP-27 expression were also reported in 20 enucleated eyes with uveal melanoma. 22 As in our study, this investigation found variability in HSP-27 staining across the uveal melanomas, but did not find any correlation between HSP-27 expression and known histopathologic prognostic parameters. We did, however, find a negative correlation between HSP-27 and the mitotic rate of the uveal melanomas. This finding will be examined in more detail in a larger study. 
Our finding of a decreased expression of HSP-27 in monosomy 3 uveal melanomas may be indirectly supported by recent studies on cutaneous melanoma. Of interest, in the human cutaneous melanoma cell line A375, 23 HSP-27 overexpression has been shown to inhibit cell proliferation and reduce cell invasiveness. Similarly, in a knockdown model of secreted protein acidic and rich in cysteines (SPARC) expression in cutaneous melanoma, 24 there is an increased HSP-27 expression, resulting in inhibited tumor progression. It could be postulated that underexpression of HSP-27 in melanoma cells results in increased tumor cell motility and invasiveness. 
Vimentin, whose gene is located on chromosome 10, is a member of the intermediate filament family of proteins, which, along with microtubules and actin microfilaments, make up the cytoskeleton of cells. Vimentin is usually expressed in malignancies of mesenchymal cell origin (e.g., melanomas) and rarely, in those tumors arising from epithelium (e.g., carcinomas). Overexpression of vimentin intermediate filaments is associated with increased tumor invasiveness and migration in a variety of tumor types in vitro, 2528 because of its role in the process of epithelial-to-mesenchymal transition. There is a complex and dynamic relationship between EMT markers, such as vimentin, smooth-muscle actin and cadherins, as well as other proteins involved in extracellular matrix remodeling and invasion (e.g., SPARC and laminin). 29  
Fuchs et al. 30 examined 52 uveal melanomas for vimentin expression, and correlated the findings with histomorphologic features and clinical course. 30 Similar to our present study, vimentin expression was found in all uveal melanomas studied, irrespective of cell type, with immunopositivity being demonstrated in more than 50% of the neoplastic cells. While Fuchs et al. could not demonstrate any relationship between vimentin expression levels and tumor size, cell type, pigmentation, or metastatic progression, we found a significantly higher vimentin expression in those uveal melanomas containing closed loops. 
One of the main strengths of our study is that in the uveal melanomas examined immunohistochemically, chromosome 3 status was determined using MLPA, which was more sensitive and informative than FISH, providing data on 13 loci on chromosome 3. 4 The relatively low sensitivity of FISH in detecting partial deletions in chromosome 3 in uveal melanoma and in subdividing these tumors into high- and low-risk metastatic subtypes, has been reported by our group 2,4 and others. 31 Despite the statistical correlation between loss of chromosome 3 and HSP-27 underexpression shown in the present study, our data give only an approximate indication of the sensitivity and specificity with which HSP-27 expression predicts monosomy 3, because of the small number of tumors examined and the relatively short follow-up period. We are currently studying a larger cohort of patients, with longer follow-up. We are particularly interested in metastatic death occurring in patients with a disomy 3 uveal melanoma showing underexpression of HSP-27 protein. 
In conclusion, our data suggest that low-to-negative HSP-27 protein expression levels in uveal melanoma are strongly associated with monosomy 3. Our preliminary results must be validated by larger studies to determine whether measurement of HSP-27 expression by immunohistochemistry can reliably predict survival when cytogenetic studies are not possible. Additional investigations are also needed to understand the functional significance of HSP-27 in uveal melanoma and its relationship to other proteins implicated in the process of epithelial-to-mesenchymal transition. 
Footnotes
 Supported by research funding from The Danish Eye Health Society, The Danish Eye Research Foundation, The Danish Medical Research Council, the John and Birthe Meyer Foundation, the Aarhus University Research Foundation, the Synoptik Foundation, the Beckett Foundation, Foreningen Østifterne, and the Eye Tumour Research Fund, Royal Liverpool University Hospital.
Footnotes
 Disclosure: S.E. Coupland, None; H. Vorum, None; N. Mandal, None; H. Kalirai, None; B. Honoré, None; S.F. Urbak, None; S.L. Lake, None; J. Dopierala, None; B. Damato, None
The authors thank Inge Kjærgaard and Mona Britt Hansen for expert technical assistance. 
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Figure 1.
 
Two-dimensional polyacrylamide gel analysis. Histograms show the densitometry measurements of the significantly up- or downregulated spots. Blue: disomy 3; red: monosomy 3.
Figure 1.
 
Two-dimensional polyacrylamide gel analysis. Histograms show the densitometry measurements of the significantly up- or downregulated spots. Blue: disomy 3; red: monosomy 3.
Figure 2.
 
Western blot analysis of uveal melanoma tissue: three tumors with disomy 3 and four tumors with monosomy 3. (A) Blots were incubated with commercial antibodies against the appropriate antigens. β-Actin was used as a loading control. (B) Mean densitometry (optical density × area) of the antibody–antigen reaction shown on the blots. Student's two-tailed t-test: *Only the two strongest visible reactions in the PDHB disomy 3 were used in the calculations.
Figure 2.
 
Western blot analysis of uveal melanoma tissue: three tumors with disomy 3 and four tumors with monosomy 3. (A) Blots were incubated with commercial antibodies against the appropriate antigens. β-Actin was used as a loading control. (B) Mean densitometry (optical density × area) of the antibody–antigen reaction shown on the blots. Student's two-tailed t-test: *Only the two strongest visible reactions in the PDHB disomy 3 were used in the calculations.
Figure 3.
 
HSP-27 immunoreactivity in FFPE uveal melanomas. (A) The tumor cells, which are of the epithelioid type, demonstrated a strong and homogenous positivity for HSP-27. (BD) Heterogeneous immunoreactivity for HSP-27 in uveal melanomas, depending on cell type, admixed reactive cells, and distance from blood vessels; (B) mixed-cell uveal melanoma; (C) epithelioid cell type uveal melanoma with admixed melanomacrophages; and (D) spindle cell type uveal melanoma. Magnification: (A) ×40 objective; (B, D) ×20 objective; (B inset; C) ×60 objective.
Figure 3.
 
HSP-27 immunoreactivity in FFPE uveal melanomas. (A) The tumor cells, which are of the epithelioid type, demonstrated a strong and homogenous positivity for HSP-27. (BD) Heterogeneous immunoreactivity for HSP-27 in uveal melanomas, depending on cell type, admixed reactive cells, and distance from blood vessels; (B) mixed-cell uveal melanoma; (C) epithelioid cell type uveal melanoma with admixed melanomacrophages; and (D) spindle cell type uveal melanoma. Magnification: (A) ×40 objective; (B, D) ×20 objective; (B inset; C) ×60 objective.
Figure 4.
 
Vimentin immunoreactivity in FFPE uveal melanomas. (A) The melanoma cells, which are of the spindle type, demonstrated a strong immunoreactivity for vimentin. (B, C) Heterogenous immunoreactivity for vimentin was present in these uveal melanomas (of epithelioid and mixed cell type, respectively), depending on cell type, the presence/absence of closed loops (inset, PAS without counterstain), and the distance from blood vessels. Magnification: (A, B inset, C) ×20 objective; (B) ×40 objective.
Figure 4.
 
Vimentin immunoreactivity in FFPE uveal melanomas. (A) The melanoma cells, which are of the spindle type, demonstrated a strong immunoreactivity for vimentin. (B, C) Heterogenous immunoreactivity for vimentin was present in these uveal melanomas (of epithelioid and mixed cell type, respectively), depending on cell type, the presence/absence of closed loops (inset, PAS without counterstain), and the distance from blood vessels. Magnification: (A, B inset, C) ×20 objective; (B) ×40 objective.
Figure 5.
 
Boxplots showing the relationship between chromosome 3 status and (A) the HSP-27 protein expression score (B) the percentage of tumor cells that stained positively for HSP-27. Probabilities show the statistical significance of the differences between monosomy 3 (M3; n = 20) and disomy 3 (D3; n = 21) uveal melanomas, as determined by Student's t-test.
Figure 5.
 
Boxplots showing the relationship between chromosome 3 status and (A) the HSP-27 protein expression score (B) the percentage of tumor cells that stained positively for HSP-27. Probabilities show the statistical significance of the differences between monosomy 3 (M3; n = 20) and disomy 3 (D3; n = 21) uveal melanomas, as determined by Student's t-test.
Table 1.
 
Commercial Antibodies Used in Western Blot Analysis
Table 1.
 
Commercial Antibodies Used in Western Blot Analysis
Antibody Name Clone/Peptide Species Manufacturer Antibody Concentration
Heat shock protein 27 Polyclonal; C terminus Goat Santa Cruz Biotechnology, Santa Cruz, CA 1:100
Vimentin Polyclonal; C terminus Rabbit Santa Cruz Biotechnology 1:100
Pyruvate dehydrogenase β Monoclonal; partial recombinant Mouse Abnova Corp., Taipei, Taiwan 1:100
β-Actin Polyclonal; aa 151–320 Chicken Abcam, Cambridge, UK 1:500
Glyceraldehyde-3-phosphate dehydrogenase Polyclonal; internal Goat Santa Cruz Biotechnology Inc. *
Glyceraldehyde-3-phosphate dehydrogenase Polyclonal; C terminus Rabbit GenWay Biotech Inc., San Diego, CA *
Fumarate hydratase Polyclonal; recombinant Rabbit Proteintech Group Inc., Chicago, IL *
α-Tubulin Polyclonal; N terminus Goat Santa Cruz Biotechnology *
Tropomyosin Polyclonal; aa 1-284 Rabbit Santa Cruz Biotechnology *
Lysozyme C Polyclonal; Internal Goat Santa Cruz Biotechnology *
F-actin capping protein α subunit (CAPZA 1) Polyclonal; aa 1-286 Chicken Abcam *
Table 2.
 
Clinicopathologic Data of Patients from the Proteomic Analysis
Table 2.
 
Clinicopathologic Data of Patients from the Proteomic Analysis
Sample ID Sex Age (y) Cell Type LBD (mm) Height (mm) Mitosis (no./40 HPF) Closed Loops Chromosome 3 Status*
IOM001 F 56 E 15.8 13.7 1 N D
IOM002 F 30 E 15.9 10.1 9 N D
IOM006 F 81 E 11.4 7.6 2 N D
IOM003 M 70 S 19.2 9.2 7 Y M
IOM004 F 42 E 19.2 12.7 28 N M
IOM005 F 78 E 20.0 9.7 2 Y M
IOM007 M 49 E 17.5 7.5 6 N M
Table 3.
 
Mass Spectrometric Analysis of Differentially Expressed Protein Spots
Table 3.
 
Mass Spectrometric Analysis of Differentially Expressed Protein Spots
Spot M3/D3 Ratio ID MW Score
0306 3.08 Tropomyosin α-4 chain TPM4_HUMAN 28.5 551
Vimentin VIME_HUMAN 53.6 343
Lysozyme C LYSC_HUMAN 16.5 75
3410 0.53 Tubulin TBAK_HUMAN 50.1 526
Pyruvate dehydrogenase El component β subunit (mitochondrial) ODPB_HUMAN 39.2 126
F-capping protein subunit α-1 CAZA1_HUMAN 32.9 36
5213 0.47 Heat-shock protein β-1 (HSP 27) HSPB1_HUMAN 22.8 572
8006 2.17 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) G3P_HUMAN 36.0 164
Fumarate hydratase (mitochondrial) FUMH_HUMAN 54.6 41
Table 4.
 
Western Blot Densitometry
Table 4.
 
Western Blot Densitometry
Antibody Disomy 3 Mean Volume (ODxArea) Monosomy 3 Mean Volume (ODxArea) P (Student's t-test) Factor Up-/Down- regulation Monosomy 3
Vimentin 5.82 12.20 0.005 X2.1 UP
HSP-27 10.51 6.67 0.003 X1.6 DOWN
PDHB 3.31* 2.53 0.097 X1.3 DOWN
Table 5.
 
Clinicopathologic Parameters and HSP-27 and Vimentin Protein Expression Data
Table 5.
 
Clinicopathologic Parameters and HSP-27 and Vimentin Protein Expression Data
Sample ID Age at Primary Diagnosis (y) Cell Type LBD (mm) Height (mm) Mitotic Frequency (No./40 HPF) Closed Loops Chromosome 3 Status (MLPA) HSP-27 Vimentin
% Tumor Cells Stained Intensity of Tumor Cells Stained Staining Score % Tumor Cells Stained Intensity of Tumor Cells Stained Staining Score
1 44 S 17.3 11.0 2 N D 20 Moderate 2 55 Weak 3
2 72 S 12.2 11.1 14 N D 15 Moderate 2 50 Moderate 6
3 80 S 15.1 10.7 2 Y D 90 Moderate 8 40 Moderate 8
4 63 S 16.1 7.5 7 Y D 55 Moderate 6 65 Moderate 6
5 69 S 12.0 3.7 3 N D 100 Moderate 8 10 Moderate 2
6 62 E 14.4 6.4 4 Y D 90 Strong 12 90 Strong 12
7 52 S 21.2 10.8 4 Y D 40 Moderate 4 85 Strong 4
8 40 S 17.5 7.9 7 N D 60 Moderate 6 90 Weak 4
9 74 S 7.8 1.9 1 N D 100 Moderate 8 90 Weak 4
10 81 S 15.3 9.5 4 N D 50 Moderate 6 75 Weak 4
11 42 S 16.7 9.1 1 N D 65 Strong 9 80 Weak 4
12 34 S 9.6 6.9 7 N D 90 Moderate 8 95 Moderate 8
13 80 S 15.1 10.7 2 Y D 95 Strong 12 95 Moderate 8
14 53 S 18.5 13.0 2 N D 75 Moderate 8 50 Moderate 6
15 67 S 17.0 9.2 2 Y D 100 Strong 12 70 Moderate 6
16 67 S 14.9 10.5 6 N D 85 Moderate 8 100 Weak 4
17 80 S 20.6 15.7 15 Y D 90 Moderate 8 95 Weak 4
18 49 S 18.3 11.7 1 N D 95 Moderate 8 60 Moderate 6
19 60 S 14.8 3.1 4 Y D 95 Moderate 8 75 Weak 4
20 46 S 12.3 4.5 4 Y D 100 Moderate 8 100 Strong 12
21 62 S 11.1 4.0 2 N D 100 Strong 12 95 Strong 12
22 67 E 22.1 10.2 31 N M 75 Moderate 8 40 Weak 2
23 62 E 19.1 8.4 7 Y M 5 Weak 1 90 Moderate 8
24 66 E 16.4 6.6 8 Y M 10 Weak 1 85 Weak 4
25 59 E 20.0 7.5 6 Y M 10 Moderate 2 95 Moderate 8
26 63 E 17.5 10.3 13 N M 15 Moderate 2 30 Moderate 4
27 44 E 13.6 6.7 7 Y M 0 Nonee 0 85 Moderate 8
28 62 E 19.1 6.8 15 Y M 75 Moderate 8 95 Moderate 8
29 48 S 15.6 8.1 9 Y M 0 Nonee 0 95 Weak 4
30 68 E 17.3 11.7 10 N M 65 Weak 3 45 Weak 2
31 71 S 17.2 12.6 7 N M 15 Moderate 2 65 Moderate 6
32 77 S 15.2 9.7 4 N M 30 Moderate 4 80 Moderate 8
33 53 S 15.2 9.7 2 Y M 85 Weak 4 80 Moderate 8
34 61 E 16.3 7.2 11 Y M 55 Moderate 6 95 Weak 4
35 53 E 20.0 8.3 12 Y M 25 Weak 2 85 Moderate 8
36 63 S 17.3 10.3 2 N M 85 Moderate 8 90 Weak 4
37 73 E 14.8 7.7 3 Y M 95 Strong 12 90 Moderate 8
38 53 S 16.6 13.6 4 N M 85 Moderate 8 80 Weak 4
39 64 E 19.6 9.8 17 Y M 85 Moderate 8 90 Weak 4
40 62 S 17.4 7.3 2 N M 90 Moderate 8 50 Weak 3
41 50 E 14.5 7.5 14 Y M 100 Strong 12 95 Moderate 8
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