G.N.P. van Muijen

Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis.

Emergence of α5β1 fibronectin- and αvβ3 vitronectin-receptor expression in melanocytic tumour progression

Cell adhesion is crucial in the process of tumour progression. As integrins are important receptor molecules involved in cell adhesion, we studied the distribution of the α1‐6, αv, αIIb, β1, β3, and β4 integrin subunits in tissue sections of common naevocellular naevi (n=22), dysplastic naevi (16), thin (24) and thick primary cutaneous melanomas (28), and melanoma metastases (25). We found correlated expression of α1/α2, of α4/α5/β3, and of α6/β4. Decrease of α6 and β4, and increase of α4 and αv were found to be correlated with melanoma progression. Furthermore, expression of α5 and β3 was detected only in primary melanoma and melanoma metastasis. Our findings indicate that during melanoma progression alterations in integrin expression occur, the most striking being emergence of α5β1 fibronectin and αvβ3 vitonectin receptor.

E-cadherin expression in human melanoma.

Loss of expression of E-cadherin, the major cell-cell adhesion receptor on keratinocytes, has been linked to tumour progression in various carcinomas. As E-cadherin has been reported to be expressed in cultured human melanocytes, we questioned whether loss of E-cadherin expression may also be related to melanocytic tumour progression. Flowcytometrical analysis demonstrated that E-cadherin was expressed on cultured normal melanocytes and naevus cells. Two non-invasive, non-metastatic melanoma cell lines showed low expression, and four invasive, metastatic melanoma cell lines did not express E-cadherin. Immunohistochemistry on frozen sections of human melanocytic lesions resulted in diffuse staining of 1/23 common naevocellular naevi and 1/13 dysplastic naevi, with no staining in any of seven early primary melanomas (≤ 1.5 mm). Staining was observed in 4/20 advanced primary melanomas (>1.5 mm) and 5/35 melanoma metastases. Thus, even though E-cadherin is expressed in cultured melanocytes and naevus cells and lost in invasive, metastatic melanoma cells in vitro, it is rarely found in early stages of melanocytic tumour progression in situ and, surprisingly, some expression can be observed in 10–20% of lesions of advanced stages.

Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR.

SummaryIn recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1–3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1–4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.

Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential.

We studied the relation between tumour vascular density and tumour growth rate, metastatic incidence and vascular permeability factor (VPF) mRNA levels in a human xenograft model described previously. Vascular density was determined by automated image analysis. Xenografts derived from cell lines BLM and MV3 showed the highest mean vascular density (MVD), the highest in vivo growth rate, high VPF mRNA levels and rapid development of lung metastases. Xenografts of cell lines M14, Mel57 and MV1 showed a significantly lower degree of vascularization, lower in vivo growth rates and lower levels of VPF mRNA, but formed lung metastases with a similar incidence as those of BLM and MV3. Xenografts from cell line 1F6 did not form lung metastases, whereas tumours derived from a spontaneous mutant of 1F6, designated 1F6m, gave rise to lung metastases to the same extent as Mel57, M14 and MV1 tumours. MVD values in 1F6 and 1F6m xenografts, VPF mRNA levels and in vivo growth rates of 1F6 and 1 F6m xenografts, however, were similar. In conclusion, in the melanoma xenograft model vascular density is correlated with in vivo growth rate and with in vitro VPF mRNA levels, but not with the ability to metastasize.

Limitations of cytokeratin 20 RT-PCR to detect disseminated tumour cells in blood and bone marrow of patients with colorectal cancer: expression in controls and downregulation in tumour tissue

Aims: Despite informative staging of patients with colorectal cancer, some patients with localised disease at diagnosis will develop recurrence or metastasis. Attempts to improve staging include sensitive detection of disseminated tumour cells in blood and bone marrow by reverse transcriptase polymerase chain reaction (RT-PCR). The results of this study have been considered in relation to the controversial results in the literature to elucidate the usefulness of cytokeratin 20 (CK20) RT-PCR to detect disseminated tumour cells further. Patients/Methods: Blood and bone marrow samples from 30 patients with colorectal cancer were studied by CK20 RT-PCR. Specificity was evaluated in 47 blood and 15 bone marrow samples from non-cancer controls. In addition, the expression of CK20 mRNA and protein was studied in normal and tumour colon tissue samples. Results: CK20 expression was detected in nine of 30 and nine of 19 of the blood and bone marrow samples from patients with colorectal cancer, respectively. In non-cancer control blood and bone marrow samples, CK20 expression was detected in 10 of 47 and four of 15, respectively. A difference between patient and control samples may be observed in terms of frequency of positive PCR tests. In tissue samples, CK20 mRNA expression was downregulated in tumour compared with normal colon tissue. Conclusions: CK20 expression was downregulated in tumour tissue compared with normal colon and a background expression of CK20 was seen in some control blood and bone marrow samples. Despite a lack of standardisation (which hampers comparison of studies), these results, together with other reports in the literature, suggest that CK20 may still be a suitable marker, but that background expression and threshold setting should be studied further.

High expression of immunotherapy candidate proteins gp100, MART-1, tyrosinase and TRP-1 in uveal melanoma.

In the treatment of cutaneous melanoma, provisional therapeutic strategies have been designed to combat tumour load using T cells that are sensitized with peptides derived from melanoma autoantigens, such as glycoprotein 100 (gp100), melanoma antigen recognized by T cells 1 (MART-1 or MelanA), tyrosinase and tyrosinase-related protein 1 (TRP-1). We recently found that gp100, MART-1 and tyrosinase are heterogeneously expressed in human cutaneous melanoma (De Vries et al (1997) Cancer Res 57: 3223-3229). Here, we extended our investigations on expression of these immunotherapy candidate proteins to uveal melanoma lesions. Cryostat sections from 11 spindle-type, 21 mixed and epithelioid tumours and four metastasis lesions were stained with antibodies specifically recognizing gp100, MART-1, tyrosinase and TRP-1. In addition, we used the DOPA reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results. High expression of gp100, MART-1 and tyrosinase was found in the uveal melanoma lesions: 80% of the lesions displayed 75-100% positive tumour cells. TRP-1 positivity was slightly less: approximately 65% of the lesions stained in the 75-100% positive tumour cell category. All uveal melanoma lesions were positive for the four markers studied, this being in contrast to cutaneous melanoma where 17% of the advanced primary lesions and metastases were negative. The presence of these antigens was a little lower in metastases. We conclude that uveal melanomas and their metastases express melanocyte-lineage immunotherapy candidate proteins very abundantly. Uveal melanomas differ in this respect from cutaneous melanoma, in which the expression of these immunotherapy antigens was much more heterogeneous. This makes uveal melanoma a suitable candidate tumour for immunotherapeutic approaches.

The Plasminogen Activation System in Tumour Invasion and Metastasis

The involvement of proteases in the metastatic spread of tumour cells and in tumour related processes, such as angiogenesis and ulceration, has been known for many decades. This chapter reviews the involvement of one proteolytic system--the plasminogen activation system--in tumour progression. In recent years, many biochemical properties of the various components of the plasminogen activation system have become known. These properties and the functional relationship between the components are discussed in the first section. Since interfering with proteolysis by tumour cells and by newly formed endothelial cells can be an objective for future therapy, experimental tumour models have been used to study the effects of inhibitors of plasminogen activation. The second section deals with this issue. Finally, the presence of the various components of the plasminogen activation system in human tumours is reviewed. Following the availability of specific ELISAs, antibodies and molecular probes, the content and the cellular distribution of the components of the plasminogen activation system have recently been mapped in various human tumours.

High tPA-expression in primary melanoma of the limb correlates with good prognosis

To investigate whether the course of primary melanoma disease correlates with expression of the various components of the proteolytic plasminogen activation (PA) system, immunohistochemical stainings for activators of plasminogen (tissue type (tPA) and urokinase type (uPA)), inhibitors of plasminogen activation (type 1 (PAI-1) and type 2 (PAI-2)) and the receptor for uPA (uPAR) were performed on 214 routinely processed melanoma lesions. All lesions were primary cutaneous melanomas, minimally 1.5u2009mm thick, and derived from patients with only local disease at the moment of diagnosis (clinically stage II (T3–4N0M0), American Joint Committee on Cancer). Median patient follow-up was 6.1 years. Single variables as immunohistochemical staining results (extent of tumour cell staining, pattern of tumour cell staining and for some components also staining of stromal cells), histopathological and clinical parameters as well as treatment variables were analysed in order to assess their prognostic importance, in terms of time to recurrence, time to distant metastasis and duration of survival. The extent of tPA tumour cell positivity, categorized as 0–5%, 6–50% and 51–100%, appeared to be of importance for these end-points. Lesions with 51–100% tPA-positive tumour cells were found to have the best prognosis, whereas lesions with 6–50% tPA-positive tumour cells had the worst. Moreover, the prognostic significance of Breslow thickness, microscopic ulceration and sex was confirmed in this study. Multivariate analyses, incorporating these relevant factors, showed that the extent of tPA tumour cell positivity was an independent prognostic factor for distant metastasis-free interval (P= 0.012) and for the duration of survival (P= 0.043).

Tetranectin and plasmin/plasminogen are similarly distributed at the invasive front of cutaneous melanoma lesions

The induction of expression of the components of the proteolytic plasminogen activation system in cutaneous melanocytic tumour progression has previously been reported. Plasminogen activators, their inhibitors, and the receptor for urokinase were present only in advanced primary melanomas and melanoma metastases. The present study reports on the presence of tetranectin and plasmin/plasminogen, two proteins connected with plasminogen activation, in cutaneous melanocytic lesions. The distribution of tetranectin and plasminogen was studied by immunohistochemistry in 105 freshly frozen melanocytic lesions of common naevocellular naevi (n=24), atypical naevi (n=14), early (n=12) and advanced (n=20) primary melanomas, and melanoma metastases (n=35). Both tetranectin and plasminogen were detected in a variety of tissue components. In all stages of melanocytic tumour progression, tetranectin was found in endothelium, perivascular dendritic cells, and leukocytes. Plasminogen was present in endothelium and in the basal layer of the normal skin. Tetranectin and plasminogen staining of fibroblastic cells at the invasive front and of extracellular matrix was, however, restricted to malignant lesions. Co‐localization of tetranectin and plasminogen was found in 50 per cent of the early primary melanomas and in more than 75 per cent of the advanced melanomas and melanoma metastases. These results suggest a coordinated role for tetranectin and plasminogen at the invasive front of melanomas. Tetranectin‐bound plasminogen may facilitate the migration of tumour cells.