Meri-Sisko Vuoristo

High Serum Levels of Matrix Metalloproteinase-9 and Matrix Metalloproteinase-1 Are Associated with Rapid Progression in Patients with Metastatic Melanoma

Purpose: Matrix metalloproteinases (MMP) are proteolytic enzymes that play an important role in various aspects of cancer progression. In the present work, we have studied the prognostic significance of serum levels of gelatinase B (MMP-9), collagenase-1 (MMP-1), and collagenase-3 (MMP-13) in patients with advanced melanoma. Experimental Design: Total pretreatment serum levels of MMP-9 in 71 patients and MMP-1 and MMP-13 in 48 patients were determined by an assay system based on ELISA. Total MMP levels were also assessed in eight healthy controls. The active and latent forms of MMPs were defined by using Western blot analysis and gelatin zymography. Results: Patients with high serum levels of MMP-9 (≥376.6 ng/mL; n = 19) had significantly poorer overall survival (OS) than patients with lower serum MMP-9 levels (n = 52; median OS, 29.1 versus 45.2 months; P = 0.033). High MMP-9 levels were also associated with visceral or bone metastasis (P = 0.027), elevated serum alkaline phosphatase level (P = 0.0009), and presence of liver metastases (P = 0.032). Serum levels of MMP-1 and MMP-13 did not correlate with OS. MMP-1 and MMP-9 were found mainly in latent forms in serum, whereas the majority of MMP-13 in serum was active (48 kDa) form. MMP-13 was found more often in active form in patients (mean, 99% of the total MMP-13 level) than in controls (mean, 84% of the total MMP-13 level; P < 0.0001). After initiating the therapy, patients with elevated levels of MMP-1 (≥29.8 ng/mL, n = 10) progressed more rapidly than patients with lower levels (median, 1.9 versus 3.5 months; P = 0.023). Serum levels of MMP-9 and MMP-13 did not correlate with the time to progression (TTP). In multivariate analysis with age and gender, MMP-9 or MMP-1 turned out to be independent prognostic factors for OS [P = 0.039; hazard ratio (HR), 1.8; 95% confidence interval (95% CI), 1.03-3.3] or TTP (P = 0.023; HR, 2.7; 95% CI, 1.15-6.4), respectively. Conclusions: Our findings provide evidence that MMP-1, MMP-9, and MMP-13 play important roles at different phases of metastatic melanoma spread and that serum MMP-9, in particular, could have clinical value in identifying patients at high risk for melanoma progression.

A phase Ii trial of bevacizumab with dacarbazine and daily low-dose interferon-α2a as first line treatment in metastatic melanoma

Metastatic melanomas are hypervascular tumours with poor prognosis. We hypothesized that treatment of metastatic melanoma with a combination of bevacizumab, a monoclonal antibody against vascular endothelial growth factor, dacarbazine (DTIC) and low-dose interferon α-2a (IFN-α2a) might lead to a synergistic inhibition of angiogenesis and regression of tumours. Patients with metastatic melanoma were treated with bevacizumab (5 mg/kg every 2 weeks), DTIC (200 mg/m2 days 1–5 every 4 weeks) and IFN-α2a (three MIU subcutaneously daily from day 15 onwards). Patients exhibiting response or stable disease after 6 months were treated with bevacizumab±IFN-α2a until disease progression. The primary study objectives were progression-free survival (PFS), overall survival and safety. Twenty-six patients were accrued. Response rate was 23% (two complete responses, four partial responses), and six patients showed stable disease. The median PFS for all patients was 2.3 months and for responders 8.1 months. The median overall survival for all patients was 11.5 months. Four life-threatening adverse events were seen: two pulmonary thromboembolisms, an intracerebral haemorrhage, and one grade 4 hypertension. One of the pulmonary emboli and the intracerebral haemorrhage were observed ≥3 months after the last bevacizumab–DTIC dose. Serum matrix metalloproteinase-9 and vascular endothelial growth factor levels changed during therapy. There was a trend towards favourable PFS among patients with only minimal or moderate change in these marker expression levels. The present regimen was active in this patient group but was also associated with remarkable vascular events.

Serum adhesion molecules and interleukin-2 receptor as markers of tumour load and prognosis in advanced cutaneous melanoma

Cell adhesion molecules are cell surface glycoproteins that may act as mediators in the metastatic process. Soluble interleukin-2 receptor (sIL-2R) is an immunological marker that may also serve as an indicator of tumour progression. Normal and neoplastic cells are capable of releasing these molecules into circulation. We evaluated the association between pretreatment serum levels of soluble intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1) and sIL-2R and metastases and survival in 50 patients with advanced melanoma. The patients with liver and/or bone metastases had significantly higher sICAM-1 levels than those with soft tissue and/or lung involvement (P=0.002). In addition, there was a trend towards higher sIL-2R levels in patients with more metastatic sites (P=0.07). In univariate analysis, the number of metastatic sites (P=0.0001, odds ratio (OR) 3.0, 95% confidence interval (CI): 1.7-5.3), the metastatic site (P=0.01, OR 2.3, 95% CI: 1.2-4.4) and the levels of sICAM-1 (P=0.011, OR 2.5, 95% CI: 1.2-5.0), sVCAM-1 (P=0.036, OR 2.1, 95% CI: 1.0-4.3) and sIL-2R (P=0.0016, OR 3.0, 95% CI: 1.5-6.0) were found to be statistically significant prognostic factors for survival. In multivariate analysis, the number of metastatic sites was the dominant prognostic indicator. After it was excluded from the analysis, the sIL-2R level and the metastatic site were found to be significant. It can be concluded, that high sICAM-1 levels suggest liver metastases and sIL-2R seems to serve as a marker of tumour load in metastatic melanoma. Furthermore, the sIL-2R level appears to add to clinical data predicting the patients outcome.

Serum VEGF-C is associated with metastatic site in patients with malignant melanoma

Vascular endothelial growth factor-C (VEGF-C) is involved in lymphatic metastatic spread. Metastatic site is a prognostic factor in melanoma. We assessed whether serum levels of VEGF-C are associated with metastatic sites or prognosis in patients treated for stage IV melanoma. The study included 64 patients, who received dacarbazine or four-drug chemotherapy (dacarbazine, vincristine, bleomycin and lomustine; BOLD) both combined with interferon-alfa. Serum samples for VEGF-C were analyzed by ELISA. The patients (n =22) with only skin and subcutaneous metastases had significantly lower mean VEGF-C levels (1 643 pg/ml) then the patients (n =42) with other distant metastases (2 584 pg/ml, Mann-Whitney, p =0.033). VEGF-C levels above the median (1 590 pg/ml) were significantly related to deep lymph node involvement (OR 3.763; 95% CI 1.038 − 13.646, p =0.034). There were no other significant associations between VEGF-C levels and tumour burden, nor were the levels significantly related to the response to therapy or survival. Those eight patients, who had received previous adjuvant IFN-alfa therapy had lower mean VEGF-C levels (1 738 pg/ml) as compared to those 56 patients without previous IFN-alfa therapy (2 335 pg/ml, ANOVA, p =0.026). This is the first study exploring serum VEGF-C in melanoma. VEGF-C might be involved in the deep lymphatic dissemination and progression of melanoma metastasis.

Randomized trial of dacarbazine versus bleomycin, vincristine, lomustine and dacarbazine (BOLD) chemotherapy combined with natural or recombinant interferon-alpha in patients with advanced melanoma.

This randomized phase II study was designed to compare the efficacy and tolerability of dacarbazine (DTIC) and bleomycin, vincristine, lomustine and DTIC (BOLD) combined with natural interferon-&agr; (nIFN-&agr;) or recombinant interferon-&agr;2b (rIFN-&agr;2b) in patients with advanced melanoma. The treatment arms were: A, DTIC plus nIFN-&agr;; B, BOLD plus nIFN-&agr;; C, DTIC plus rIFN-&agr;2b; D, BOLD plus rIFN-&agr;2b. One hundred and eight patients were randomized, of whom 106 were eligible to be analysed for efficacy. Overall, 56% of patients had abdominal visceral and/or bone involvement. The response rates were 8% (2/25) in arm A, 13% (4/31) in arm B, 12% (3/25) in arm C and 24% (6/25) in arm D. The differences were not statistically significant by the usual chi-squared test. However, when analysed using the Cochran–Armitage trend test, the one-sided P values were close to significant (0.085 and 0.033). All of the eight complete responses occurred in patients with soft tissue and/or lung metastases and the BOLD regimens produced six of them. There were no significant differences in survival (arm A, 11.1 months; arm B, 9.8 months; arm C, 9.1 months; arm D, 7.5 months; P=0.62). BOLD was more toxic than DTIC. With the present sample size, there were no statistically significant differences in efficacy between the arms, but there was a trend towards a higher response rate with BOLD plus rIFN-&agr;2b. Patients with soft tissue or lung metastases may achieve more complete responses with BOLD regimens.

The value of serum S-100beta and interleukins as tumour markers in advanced melanoma.

&NA; Recently serum S‐100&bgr; has shown promise as a tumour marker in melanoma; however, its use as a prognostic marker in the advanced stage needs to be confirmed. Interleukins (ILs) may mediate regression or progression of cancer. in order to study their relation to the metastatic profile and survival, we evaluated the association between pretreatment serum levels of S‐100&bgr;, IL‐6, IL‐10 and IL‐12 and metastatic site and survival in 50 patients with advanced melanoma who were to receive chemoimmunotherapy. Patients with liver and/or bone metastases had significantly higher median concentrations of S‐100&bgr;, IL‐6 and IL‐10 than those with only skin, nodal and/or lung involvement. The differences in IL‐12 levels were unremarkable. Using univariate analysis, the S‐100&bgr; level and metastatic profile were found to be statistically significant prognostic factors for survival. Using multivariate analysis the S‐100&bgr; level was the most powerful prognostic indicator, while the metastatic profile was found to be significant after exclusion of S‐100&bgr;. The findings suggest that elevated serum levels of S‐100&bgr;, IL‐6 and IL‐10 reflect concurrent liver or bone metastases in melanoma. S‐100&bgr; is also an independent prognostic marker. Pretreatment IL levels were not associated with outcome.

Serum Matrix Metalloproteinase-2 as a Prognostic Marker in Advanced Cutaneous Melanoma

The association was studied between serum concentration of matrix metalloproteinase-2 (MMP-2) and metastatic site, survival and disease progression in patients with advanced cutaneous melanoma. The patient population consisted of 50 patients who were treated with chemoimmunotherapy. The median baseline serum concentration of MMP-2 was 724 ng/ml (range 500-2297 ng/ml). There were no significant differences in MMP-2 levels according to metastatic site. Baseline MMP-2 concentration did not have a prognostic value. The patients with levels below 800 ng/ml survived for 8.8 months and those with higher levels for 9.7 months. On serial measurements, median serum MMP-2 concentration at disease progression in 25 patients was significantly higher than before treatment. Only five samples at response were available, and the levels were not significantly different from baseline levels. In conclusion, serum MMP-2 is not a prognostic marker in advanced melanoma. It also appears to be of limited clinical value in monitoring.The association was studied between serum concentration of matrix metalloproteinase-2 (MMP-2) and metastatic site, survival and disease progression in patients with advanced cutaneous melanoma. The patient population consisted of 50 patients who were treated with chemoimmunotherapy. The median baseline serum concentration of MMP-2 was 724 ng/ml (range 500-2,297 ng/ml). There were no significant differences in MMP-2 levels according to metastatic site. Baseline MMP-2 concentration did not have a prognostic value. The patients with levels below 800 ng/ml survived for 8.8 months and those with higher levels for 9.7 months. On serial measurements, median serum MMP-2 concentration at disease progression in 25 patients was significantly higher than before treatment. Only five samples at response were available, and the levels were not significantly different from baseline levels. In conclusion, serum MMP-2 is not a prognostic marker in advanced melanoma. It also appears to be of limited clinical value in monitoring.

Intermittent interferon and polychemotherapy in metastatic melanoma

This study was conducted to evaluate the efficacy and the tolerability of a four-drug chemotherapy regimen combined with interferon α(IFN) in metastatic melanoma. Between March 1991 and August 1993, 55 patients with advanced melanoma were enrolled for the present multicentre phase II study. Fortynine patients were eligible and evaluable for toxicity; 48 patients were evaluable for response. The treatment schedule consisted of a 5-day regimen of dacarbazine, vincristine, bleomycin and lomustine, plus 6×106 IU IFNα three times weekly subcutaneously for 2 weeks starting on day 8. The cycle was repeated on day 29. Among the 48 assessable patients, 16 objective responses were seen, yielding a response rate of 33% (95% confidence interval 20%–46%). Seven patients achieved a complete response (CR) of a median of 6 + months (range 1 + to 21 + months) and 9 patients achieved a partial response (PR) of a median of 9 months (range 4–13 months). The median overall survival was 12 + months (range 6+ to 23 + months) for the patients with CR and 15 + months (range 8–20 months) for the patients with PR. Even the survival of the 7 patients with stable disease was fairly long (median 12, range 7–17 months), appearing to be significantly longer than the survival of the 25 patients with progressive disease (median 5, range 1–24 + months). The treatment was moderately well tolerated, although all patients experienced some mild form of toxicity, mostly gastrointestinal symptoms, neurotoxicity and haematotoxicity. Grade 3–4 adverse effects were noted in 39% of the patients. No toxic deaths occurred. It can be concluded that the present regimen produces meaningful responses for patients with metastatic melanoma. A randomised study is needed to determine the effect on survival.

A combination of subcutaneous recombinant interleukin-2 and recombinant interferon-α in the treatment of advanced renal cell carcinoma or melanoma

In this phase II study, we have evaluated the efficacy and toxicity of low-dose subcutaneous (s.c.) recombinant interleukin-2 (IL-2) and recombinant interferon (IFN)-alpha in 16 patients with advanced renal cell carcinoma (RCC) and in 4 patients with advanced melanoma. The complete course on this protocol comprised 6 weeks of s.c. IL-2 plus IFN-alpha followed by a 2-week rest period. The treatment was moderately strenuous for patients, requiring frequent dose reductions; only eight cycles (30%) could be administered to 75-100% of the projected dose. Main side-effects were fever, fatigue, hypotension, liver toxicity, neurotoxicity and skin reactions. Among the evaluable 17 patients, two responses (one partial, one complete) were seen in patients with RCC. This regimen proved to be rather toxic and yielded a modest response rate of 15% in RCC, but initial findings concerning the duration of survival seem promising.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay

Cancer‐testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16‐positive patients. A highly sensitive time‐resolved fluorescence‐based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon‐Mann‐Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.