Annals of Oncology Advance Access originally published online on September 13, 2006
Annals of Oncology 2006 17(12):1830-1834; doi:10.1093/annonc/mdl305
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2006 European Society for Medical Oncology
melanoma |
Increased chondroitin sulphate proteoglycan expression (B5 immunoreactivity) in metastases of uveal melanoma
1 Department of Hematology, Oncology, and Transfusion Medicine, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
2 Department of Ophthalmology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
3 TRION Research GmbH, Martinsried, Germany
4 TRION Pharma GmbH, Munich, Germany
* Correspondence to: Dr D. Nagorsen, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Medizinische Klinik III, Hematology, Oncology, and Transfusion Medicine, Hindenburgdamm 30, 12200 Berlin, Germany. Tel: +49-30-8445-2337; Fax: +49-30-8445-4468; E-mail: Dirk.Nagorsen{at}charite.de
 |
Abstract |
|---|
 Top Abstract introductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
Background: Metastatic uveal melanoma has a poor prognosis and limited therapeutic options. Proteoglycans are involved in tumor cell invasion and metastatic behavior. The mAbB5 stains a chondroitin sulphate proteoglycan (CSPG) on cutaneous melanoma cells. Here, we compare the B5-staining of CSPG in primaries and metastases of uveal melanoma.
Material and methods: Immunohistopathological staining was performed in 15 cutaneous and 39 uveal melanoma samples. A score for intracellular and surface staining was established. B5 staining was compared in primaries and metastases of uveal melanoma using Student's t-test.
Results: Eight of 11 (73%) uveal melanoma metastases were positive for B5-staining whereas only 5 of 28 (18%) primary uveal melanoma samples were B5-positive (P < 0.001). Nine of 15 cutaneous melanoma samples (60%) were B5-positive without significant difference between primary and metastatic lesions. Surface staining was found both on uveal melanoma metastases and cutaneous melanomas.
Conclusions: CSPG was expressed significantly more often in metastases than in primaries of uveal melanoma. It potentially may be one factor associated with metastatic spread. Further studies are needed to determine its use as prognostic factor. The mAbB5 may also be a promising tool for immunotherapy due to its strong staining of CSPG on the surface of cutaneous and metastatic uveal melanoma cells.
Key words: uveal melanoma, ocular melanoma, chondroitin sulphate proteoglycan, immunotherapy, immunohistochemistry
|
introduction |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
Uveal melanoma is a rare disease with an annual incidence of approximately 6/1 000 000 [1]. Primary uveal melanoma can be successfully treated by surgery and radiation. However, about 35% of the tumors will eventually metastasize – mainly to the liver – with a very poor prognosis [2]. Patients with metastatic uveal melanoma have a median survival of less than 6 months [3]. While radical surgery of metastases seems to have some effect in selected patients [4], the various chemotherapeutic regimens evaluated so far [5, 6] fail to provide adequate treatment for metastatic uveal melanoma.
Several attempts have been made to find clinical predictors or characteristic genomic patterns for the metastatic spread of uveal melanoma. Ciliary body involvement significantly increases the risk of metastases [7]. Furthermore, extraocular growth and a largest tumor diameter >14 mm are associated with a significantly lower 5-year survival [7]. Monosomy 3 has been found as an indicator of poor prognosis [8, 9]. Thus, downregulation of genes on chromosome 3, as determined by gene expression analysis, correlates adversely with survival [10]. A similar prediction was described for upregulated genes on chromosome 8 [10].
Cell surface proteoglycans, mediators of cell-cell and cell-extracellular-matrix interaction, cannot be adequately investigated by gene analysis. Chondroitin sulphate proteoglycan (CSPG) is a molecule abundantly expressed early in the progression of cutaneous melanoma [11]. Melanoma adhesion has been inhibited by mAb directed against CSPG [12], and CSPG has also been associated with melanoma invasion in vitro [13]. Mouse models have shown that transfection with NG2, the rat homolog of human melanoma CSPG, is associated with increased metastatic spread [14]. The interaction between CSPG and matrix metalloproteinase 3 may be crucial for melanoma invasion [15]. Furthermore, it has been shown that chondroitin sulphate proteoglycan may facilitate melanoma progression by activating key signaling pathways important for tumor invasion and growth [16]. Thus melanoma-associated CSPG plays an important part in melanoma cell biology with regard to invasion and metastasis and is a potential target for melanoma immunotherapy [17].
Monoclonal antibody B5 detects immunoprecipitates of Mr 250 000 and Mr >500 000 linked to CSPG [18]. Using mAbB5, Real et al. [19] found that 21 of 22 (95%) melanoma cell lines and 17 of 22 (77%) cutaneous melanoma tissues were positive for CSPG. B5 staining is restricted to melanomas, nevi, astrocytomas and cultured melanocytes. No normal or malignant tissues other than endothelial cells (in normal and malignant tissues) and keratinocytes were stained by B5 [19]. Recently, Ruf et al. [20] have constructed a trifunctional antibody compound consisting of one Fab fragment directed against CD3, one Fab against CSPG (based on B5) and an Fc fragment. This trifunctional antibody was able to mediate specific melanoma cell lysis and induce a strong Th1 cytokine release pattern.
It is not yet known whether B5 reactivity can be found in uveal melanoma or whether there is a difference between primary and metastatic uveal melanomas. We performed the present study to compare mAbB5 staining of CSPG in paraffin slides of primary and metastatic uveal melanomas. For control purposes, we also analyzed B5 reactivity in primary and metastatic cutaneous melanoma sections.
|
materials and methods |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
specimens
Twenty-eight enucleated eyes of patients with primary uveal melanoma, 11 biopsy specimens from patients with metastatic uveal melanoma, and 15 samples from patients with cutaneous melanoma (five primary, 10 metastatic) were routinely fixed and paraffin-embedded. Tissue samples were sectioned (5 µm thick). Paraffin sections routinely stained with hematoxylin and eosin were assessed for histopathology. There were no paired samples (matched primaries/metastases) in our study.
antibodies and immunohistochemistry
Mouse anti-human IgG monoclonal antibody (mAbB5) was used as the primary antibody. It was provided by Trion Research (Munich, Germany) and used in 1:50 dilution. Paraffin-embedded tissue sections were incubated for 20 minutes at 60°C, deparaffinated in xylene and graded alcohols and rehydrated in TRIS buffered saline (TBS, pH 7.5). After additional boiling in citrate buffer for 2 min, sections were incubated with mAbB5 for 30 min at room temperature. Briefly, immunoreactivity was detected by the APAAP method using the DAKOCytomation ChemMateTM detection kit APAAP, mouse, according to the manufacturer's instructions.
assessment of immunostaining and statistical analysis
Two researchers (PK and DN) independently assessed immunostained sections using a Zeiss Axioskop 40 light microscope at a final magnification of 400x. Sections were analyzed for the percentage of B5 cell positivity (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%) and graded for staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong). The grades of the two observers were averaged. For each section, a combined expression score (CES) for mAbB5 staining was obtained by multiplying the percentage by the intensity score. The combined expression score was evaluated separately for intracellular and surface staining. The mAbB5 staining was rated positive if the intracellular or surface combined expression score was 
6. A two-sided Student's t-test was used to compare the number of positive combined expression scores of B5 staining in metastases and primaries of uveal melanoma.
clinical data of patients with primary uveal melanoma
We retrospectively analyzed clinical data of all 28 patients with primary uveal melanoma. Using a one-sided Student's t-test for a post-hoc analysis we tested whether tumors with B5 positive cells (CES 2) had developed metastases more frequently than tumors without B5 staining in their primary uveal melanoma.
|
results |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
CSPG expression in primary uveal melanoma
Twenty-eight primary uveal melanoma samples were analyzed for B5 staining. Five of 28 (18%) samples were positive according to the criteria applied. None of the samples showed positive surface B5 staining. See Table 1. A representative sample is shown in Figure 1B.
|
|
CSPG expression in uveal melanoma metastases
Eleven samples of uveal melanoma metastases (mainly liver metastases) were analyzed for B5 staining. Eight of the 11 samples (73%) were positive according to the criteria applied. Four of eight positive samples (50%) showed positive surface B5 staining. See Table 2. A representative sample is shown in Figure 1A.
|
CSPG expression in cutaneous melanoma
Five primary cutaneous melanomas and 10 cutaneous melanoma metastases (mainly skin and lymph node metastases) were analyzed for B5 staining. Nine of the 15 samples (60%) were positive according to the criteria applied. There was no significant difference (P = 0.297) between primaries (four of five (80%) positive) and metastases of cutaneous melanoma (five of 10 (50%) positive). All nine positive samples also showed positive surface B5 staining. See Table 3. Representative samples are shown in Figure 1 C+D.
|
comparison of B5 staining in primaries and metastases of uveal melanoma
B5 positivity was found in eight of 11 (73%) uveal melanoma metastases but only in five of 28 (18%) primary uveal melanomas. This difference was highly significant (P < 0.001) using Student's t-test.
metastatic spread related to B5-staining
Clinical follow-up data on metastatic spread was available for 19 out of 28 (68%) patients with primary uveal melanoma samples. Mean follow-up was 60.7 months (range 3–96 months). Of these 19 patients, four had developed liver or lung metastases after a mean of 6.5 (range 4–15) months after resection of primary uveal melanoma. Three out of nine patients (33%) with B5 immunoreactivity (CES 2) in their primary uveal specimen had developed metastases whereas only one out of 10 patients (10%) without B5 immunoreactivity had developed metastases. This difference is not statistically significant (P = 0.12). See Table 1.
|
discussion |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
While B5 immunoreactivity has been previously studied in cutaneous melanoma [19], we present the first study to investigate B5 staining in uveal melanoma. With positive staining in 18% of primary uveal melanoma sections and 73% of metastatic ones, we found a significantly higher B5-determined CSPG expression in metastases than in primaries of uveal melanoma.
The CSPG detected by B5 is related to the NG2 antigen, a rat homolog of the human melanoma CSPG detected by mAb 9.2.27 [21, 22]. In a recent study, mAb 9.2.27 was used to investigate proteoglycan immunoreactivity in 26 primary uveal melanoma samples [23]. Six samples were not analyzed for technical reasons. Eighteen of the remaining 19 tumors (95%) were rated positive for mAb 9.2.27 staining. In our analyses, mAbB5-detected proteoglycan expression rates in primary uveal melanoma are lower than in the study by Li et al. [23]. This difference might be explained by two factors. Firstly, mAb 9.2.27 detects a CSPG-associated glycoprotein related to the one detected by mAbB5 [21, 22] but does not necessarily detect the same antigen as mAbB5. Secondly, in contrast to Li et al. [23], we set a higher threshold (combined expression score 6) to determine positive staining. Therefore, our conservative analysis tends to underestimate proteoglycan expression in melanomas. This assumption is also supported by the relatively low B5 staining rate of 60% for cutaneous melanoma in our study compared to 77% in an earlier study [19]. These differences do not weaken the evidence our study provides, because, even if we had lowered the threshold of the combined expression score (CES) from 6 to 4, we would still have found a significantly higher B5 staining rate in metastases than in primaries of uveal melanoma (descriptively calculated using Student's t-test: P < 0.001).
Metastatic uveal melanoma is a disease still lacking adequate treatment. Thus, target therapy using an active mAb would have a substantial therapeutic potential. Antibody-based treatments of several malignant diseases are well established in clinical practice nowadays [23]. A suitable target needs to be strongly and predominantly expressed by malignant cells. The target antigen has to be expressed on the cell surface and should preferentially be involved in tumor proliferation and growth. As pointed out earlier, only endothelial cells (in normal and malignant tissue) and keratinocytes are stained by B5. Here we found B5 staining in the majority of uveal melanoma metastases, 50% of which are stained on the cell surface. The epitope detected by B5 is part of the CSPG and may therefore play a functional role in metastatic spread and tumor proliferation. Ruf et al. [20] found that B5, as part of a trifunctional antibody (bispecifically directed against B5, CD3, and an Fc fragment), mediated specific lysis of various melanoma cell lines in relation to the level of antigen expression in cytotoxicity experiments. The B5-based trifunctional antibody induced a strong Th1 cytokine release pattern with large amounts of IFN-gamma. The staining pattern of B5 and the experimental data of Ruf et al. [20] suggest that mAbB5-based compounds might be candidates for target therapy in metastatic uveal melanoma.
To determine whether B5 immunoreactivity in primary uveal melanomas has an impact in metastatic spread, we retrospectively analyzed clinical data of patients with primary uveal melanoma. Four out of 19 patients available for analysis had developed metastases. Despite the small sample number, this corresponds well with the reported frequency of metastatic spread as mentioned above. There tend to be more metastases among patients with B5 positive primary uveal melanoma samples. However, this difference is not significant and must be interpreted with great caution due to the small number of patients. It therefore remains unclear whether B5 staining can be of prognostic value. Further studies are planned to evaluate the clinical behavior of B5-positive uveal melanomas and investigate the impact on tumor cell biology and metastatic spread.
In summary, the CSPG detected by B5-staining is expressed significantly more often in metastases than in primaries of uveal melanoma. It potentially may be one factor associated with the metastatic behavior of this tumor. Further studies are needed to determine whether it can be used as a prognostic factor. The monoclonal AbB5 strongly stains CSPG on the surface of a substantial number of cutaneous and metastatic uveal melanoma cells in situ and may thus be a promising tool for immunotherapy of melanoma.
|
Acknowledgements |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
The authors thank Dr S. E. Coupland for scientific advice and Heidrun Protz for excellent technical assistance.
Received for publication March 10, 2006. Revision received July 5, 2006. Accepted for publication July 6, 2006.
|
References |
|---|
|
TopAbstractintroductionmaterials and methodsresultsdiscussionAcknowledgementsReferences |
|---|
1. Egan KM, Seddon JM, Glynn RJ, et al. (1988) Epidemiologic aspects of uveal melanoma. Surv Ophthalmol 32:239–251.[CrossRef][ISI][Medline]
2. Diener-West M, Hawkins BS, Markowitz JA, Schachat AP. (1992) A review of mortality from choroidal melanoma. II. A meta-analysis of 5-year mortality rates following enucleation, 1966 through 1988. Arch Ophthalmol 110:245–250.[Abstract]
3. Singh AD and Borden EC. (2005) Metastatic uveal melanoma. Ophthalmol Clin North Am 18:143–150.[CrossRef][Medline]
4. Rivoire M, Kodjikian L, Baldo S, et al. (2005) Treatment of liver metastases from uveal melanoma. Ann Surg Oncol 12:422–428.
5. Schmidt-Hieber M, Schmittel A, Thiel E, Keilholz U. (2004) A phase II study of bendamustine chemotherapy as second-line treatment in metastatic uveal melanoma. Melanoma Res 14:439–442.[CrossRef][ISI][Medline]
6. Schmittel A, Scheulen ME, Bechrakis NE, et al. (2005) Phase II trial of cisplatin, gemcitabine and treosulfan in patients with metastatic uveal melanoma. Melanoma Res 15:205–207.[CrossRef][ISI][Medline]
7. Schmittel A, Bechrakis NE, Martus P, et al. (2004) Independent prognostic factors for distant metastases and survival in patients with primary uveal melanoma. Eur J Cancer 40:2389–2395.[CrossRef][ISI][Medline]
8. White VA, Chambers JD, Courtright PD, et al. (1998) Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer 83:354–359.[CrossRef][ISI][Medline]
9. Sandinha MT, Farquharson MA, Roberts F. (2004) Identification of monosomy 3 in choroidal melanoma by chromosome in situ hybridisation. Br J Ophthalmol 88:1527–1532.
10. Onken MD, Worley LA, Ehlers JP, Harbour JW. (2004) Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 64:7205–7209.
11. Reisfeld RA and Cheresh DA. (1987) Human tumor antigens. Adv Immunol 40:323–377.[ISI][Medline]
12. de Vries JE, Keizer GD, te Velde AA, et al. (1986) Characterization of melanoma-associated surface antigens involved in the adhesion and motility of human melanoma cells. Int J Cancer 38:465–473.[ISI][Medline]
13. Chattopadhyay P, Starkey J, Morrow WJ, Raychaudhuri S. (1992) Murine monoclonal anti-idiotope antibody breaks unresponsiveness and induces a specific antibody response to human melanoma-associated proteoglycan antigen in cynomolgus monkeys. Proc Natl Acad Sci U S A 89:2684–2688.
14. Burg MA, Grako KA, Stallcup WB. (1998) Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells. J Cell Physiol 177:299–312.[CrossRef][ISI][Medline]
15. Iida J, Pei D, Kang T, et al. (2001) Melanoma chondroitin sulphate proteoglycan regulates matrix metalloproteinase-dependent human melanoma invasion into type I collagen. J Biol Chem 276:18786–18794.
16. Yang J, Price MA, Neudauer CL, et al. (2004) Melanoma chondroitin sulphate proteoglycan enhances FAK and ERK activation by distinct mechanisms. J Cell Biol 165:881–891.
17. Campoli MR, Chang CC, Kageshita T, et al. (2004) Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulphate proteoglycan (MSCP) with biological and clinical significance. Crit Rev Immunol 24:267–296.[CrossRef][ISI][Medline]
18. Houghton AN, Eisinger M, Albino AP, et al. (1982) Surface antigens of melanocytes and melanomas. Markers of melanocyte differentiation and melanoma subsets. J Exp Med 156:1755–1766.
19. Real FX, Houghton AN, Albino AP, et al. (1985) Surface antigens of melanomas and melanocytes defined by mouse monoclonal antibodies: specificity analysis and comparison of antigen expression in cultured cells and tissues. Cancer Res 45:4401–4411.
20. Ruf P, Jager M, Ellwart J, et al. (2004) Two new trifunctional antibodies for the therapy of human malignant melanoma. Int J Cancer 108:725–732.[CrossRef][ISI][Medline]
21. Morgan AC Jr, Galloway DR, Reisfeld RA. (1981) Production and characterization of monoclonal antibody to a melanoma specific glycoprotein. Hybridoma 1:27–36.[Medline]
22. Bumol TF and Reisfeld RA. (1982) Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc Natl Acad Sci USA 79:1245–1249.
23. Li Y, Madigan MC, Lai K, et al. (2003) Human uveal melanoma expresses NG2 immunoreactivity. Br J Ophthalmol 87:629–632.
24. Stern M and Herrmann R. (2005) Overview of monoclonal antibodies in cancer therapy: present and promise. Crit Rev Oncol Hematol 54:11–29.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||