Original article 475
Clinical significance of serum fibronectin and vitronectin levels in melanoma patients Faruk Tas, Senem Karabulut, Elif Bilgin, Didem Tastekin and Derya Duranyildiz Fibronectin and vitronectin are the important components of the extracellular matrix proteins. The aim of this study was to determine the clinical significance of these protein serum levels in patients with melanoma. A total of 60 patients with a pathologically confirmed diagnosis of melanoma were enrolled in this study. Serum fibronectin and vitronectin concentrations were determined using the solid-phase sandwich ELISA method. Thirty age-matched and sexmatched healthy controls were included in the analysis. The baseline serum fibronectin and vitronectin levels were significantly higher in patients with melanoma than those in the healthy control group (P < 0.001 and P = 0.04, respectively). However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum lactate dehydrogenase levels, and response to chemotherapy were not found to be correlated with either serum fibronectin or vitronectin concentrations (P > 0.05).
Moreover, neither serum fibronectin nor vitronectin levels played a prognostic role in outcome in melanoma patients (P = 0.47 and 0.24, respectively). In conclusion, serum levels of both fibronectin and vitronectin may be diagnostic markers in melanoma patients. However, their predictive and prognostic values were not determined. Melanoma Res 24:475–479 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Materials and methods
Melanoma is a malignancy characterized by its highly invasive and metastatic nature, properties that require interaction of the melanoma cells with the extracellular matrix (ECM) through cell adhesion, migration, proliferation, and metastasis.
Patients
Similarly, fibronectin and vitronectin, major structural components of the basement membrane, are strong promoters of cell adhesion, migration, differentiation, and proliferation through integrins and other cell surface receptors. Increased fibronectin [1–8] and vitronectin [9–19] expressions were also detected in melanoma cell lines with increased melanoma cell proliferation and metastatic potential. Although all available findings were obtained from preclinical trials, so far, a clinical study investigating the clinical significance of these ECM markers of the plasma/serum in melanoma patients has been limited [20]. Thus, the significance of the serological levels of these ECM markers in melanoma patients is not yet known. Therefore, we evaluated the soluble serum levels of fibronectin and vitronectin in melanoma patients and assessed associations with the prognosis, various known clinical variables, and response to chemotherapy to examine whether these are potential new biomarkers for use in the treatment of melanoma in this study. 0960-8931 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Melanoma Research 2014, 24:475–479 Keywords: fibronectin, melanoma, serum, vitronectin Institute of Oncology, University of Istanbul, Istanbul, Turkey Correspondence to Faruk Tas, MD, Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey Tel: + 90 212 534 80 78; fax: + 90 212 534 80 78; e-mail: [email protected] Received 22 March 2014 Accepted 6 June 2014
This study included 60 patients admitted to the Institute of Oncology, University of Istanbul, with histologically confirmed melanoma. Advanced patients with bidimensionally measurable disease without a history of chemotherapy/radiotherapy in the last 6 months were included in the study. The staging was determined according to the American Joint Committee on Cancer (AJCC) staging system. The pretreatment evaluation included a detailed assessment of clinical history and physical examination with a series of biochemistry tests including lactate dehydrogenase (LDH) and complete blood cell counts. Those with Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less and appropriate blood chemistry tests received chemotherapy on an outpatient basis comprising interferonα, cisplatin, dacarbazine, or temozolomide compounds with/without radiotherapy depending on the stage of disease. Follow-up programs consisted of clinical, laboratory, and radiological assessments performed at 8 weeks intervals during chemotherapy or every 12 weeks for no anticancer treatment. Response to treatment was determined according to revised Response Evaluation Criteria In Solid Tumors (RECIST) criteria version 1.1. For comparison of serum levels of fibronectin and vitronectin, 30 age-matched and sex-matched healthy controls DOI: 10.1097/CMR.0000000000000107
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
476
Melanoma Research
2014, Vol 24 No 5
were included in the analysis. Informed consent was obtained from all patients and the study was reviewed and approved by our local ethical committee. Measurement of serum fibronectin and vitronectin levels
Serum samples were obtained on first admission before any adjuvant and metastatic treatment was administered or follow-up of patients. Blood samples were obtained from patients with malignant melanoma and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at − 20°C until analysis. The fibronectin ELISA (eBioscience, Vienna, Austria) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human fibronectin in samples. Serum samples, standards, and biotinconjugate were added to the wells, which were precoated with human fibronectin monoclonal antibody, and allowed to incubate for 2 h. Unbound material was washed away. Fibronectin combined with streptavidin–horseradish peroxidase were added to form an immune complex and then allowed to incubate for 1 h. Unbound material was washed away. Chromogen solution was added and incubated for about 10 min (protected from light) for the conversion of the colorless solution into a blue solution, the intensity of which was proportional to the amount of fibronectin in the sample. Because of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta, Georgia, USA) at 450 nm. The results were expressed as ng/ml. The Vitronectin ELISA (Wuhan EIAab Science, Wuhan, China) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human vitronectin in samples. Serum samples and standards were added to the wells, which were precoated with human vitronectin monoclonal antibody, and allowed to incubate for 2 h. Unbound material was washed away. Vitronectin combined with streptavidin–horseradish peroxidase were added to form an immune complex and then allowed to incubate for 1 h. Unbound material was washed away. Chromogen solution was added and incubated for 15–25 min (protected from light) for the conversion of the colorless solution into a blue solution, the intensity of which was proportional to the amount of vitronectin in the sample. Because of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer) at 450 nm. The results were expressed as ng/ml. Statistical analysis
Continuous variables were categorized using median values as the cutoff point. Assessment of relationships, comparisons between various clinical/laboratory parameters
and serum levels of fibronectin and vitronectin assays were carried out using the Mann–Whitney U-test and the Kruskal–Wallis test for two and three groups, respectively. Spearman’s rank order correlation was used for correlation analysis. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. The Kaplan–Meier method was used to estimate the survival of patients and differences in survivals were assessed using the log-rank statistics. A P value of less than 0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.
Results A total of 60 patients with stage I–II (n = 13), stage III (n = 14), stage IV (n = 31), and undetermined stage (n = 2) with a pathologically confirmed diagnosis of melanoma were enrolled in this study. The majority of the patients with metastatic disease were M1c (18/31, 58%). The baseline histopathological characteristics and the demographic characteristics of the patients are listed in Table 1. The median age at diagnosis was 53.5 years (range 16–88 years); 55% of the patients in the group were men (n = 33). The baseline serum fibronectin and vitronectin levels were significantly higher in patients with melanoma than those in the healthy control group (P < 0.001 and Table 1
Patient and disease characteristics
Variables Number of patients Age (years) < 50/ ≥ 50 Sex Male/female Site of lesion Axial/extremity/unknown Histology Nodular/non-nodular/unknown Stage of disease I–II/III/IV/unknown Tumor statusa Thin (T1–T2)/thick (T3–T4)/unknown Node statusa Negative/positive M1 status M1a + b/M1c Serum hemoglobin level (12 g/dl) Low/normal/unknown Serum WBC count (10 000) Normal/elevated/unknown Serum LDH level (450 U/l) Normal/elevated ESR (40/h) Normal/elevated/unknown Response to chemotherapyb Yes/no/unknown Last status Alive/dead
n 60 26/34 33/27 38/16/6 9/31/20 13/14/31/2 10/16/1 12/14 13/18 19/40/1 50/9/1 48/12 28/20/12 10/11/10 41/19
ESR, erythrocyte sedimentation rate; LDH, lactate dehydrogenase; WBC, white blood cell. In nonmetastatic patients. b In metastatic patients. a
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Fibronectin/vitronectin in melanoma Tas et al. 477
Values of serum extracellular matrix assay levels in melanoma patients and healthy controls
No significant relationship was found between the serum levels of fibronectin and vitronectin (rs = 0.190, n = 60, P = 0.884, Spearman’s correlation).
Patients (n = 60) Range
Controls (n = 30)
Assays
Median
Median
Range
P
Fibronectin (ng/ml) Vitronectin (ng/ml)
515 362–678 192 58–532 < 0.001 1259.3 258.3–4821.8 1111.1 33.9–2944.0 0.04
Results (median and range) of comparisons between the extracellular matrix assays and various clinical parameters in melanoma patients
Table 3
Parameters Age (P) (years) Old (≥50) Young (< 50) Sex (P) Male Female Site of lesion (P) Axial Extremity Histology (P) Non-nodular Nodular Stage of disease (P) I–II III IV Tumor status (P) Thin Thick Node status (in M0 disease) (P) Negative Positive Metastasis status (in all patients) (P) 0 1 M1 status (P) a–b c Serum hemoglobin level (P) Normal Low Serum WBC count (P) Normal Elevated ESR (P) Normal Elevated Serum LDH level (P) Normal Elevated Response to chemotherapy (P) Responsive Nonresponsive
Fibronectin (ng/ml)
Vitronectin (ng/ml)
0.43 (192–678) (362–676) 0.07 (362–678) (366–678) 0.64 (362–678) (366–674) 0.43 (376–676) (362–678) 0.95 (392–674) (362–674) (366–678) 0.42 (392–674) (362–674) 0.63 (392–674) (362–674) 0.84
0.24 1160.6 (258.3–4821.8) 1354.4 (515.6–4370.3) 0.12 1060.2 (258.3–4821.8) 1461.1 (677.6–3811.3) 0.34 1259.3 (258.3–4821.8) 1784.4 (677.6–4634.4) 0.48 1316.5 (258.3–4821.8) 974.1 (542.1–4370.3) 0.93 1060.2 (258.3–4821.8) 1320.7 (581.1–4634.4) 1316.5 (398.2–4370.3) 0.14 1646.8 (774.8–4821.8) 1017.2 (258.3–4634.4) 0.56 1056.8 (258.3–4821.8) 1320.7 (581.1–4634.4) 0.77
518 (362–676) 486 (366–678) 0.54 456 (394–678) 517 (366–676) 0.38 520 (362–678) 484 (366–678) 0.14 486 (362–678) 542 (416–676) 0.81 501 (362–678) 480 (388–678) 0.77 518 (362–678) 480 (366–676) 0.39 502 (404–678) 458 (388–676)
1180.2 (258.3–4821.8) 1316.5 (398.2–4370.3) 0.83 1316.5 (677.6–3682.1) 1316.6 (398.2–4370.3) 0.89 1259.3 (258.3–4821.8) 1320.3 (398.2–4370.3) 0.72 1283.5 (258.3–4821.8) 1271.9 (398.2–4127.5) 0.33 1461.1 (581.1–4821.8) 1175.8 (258.3–4370.3) 0.57 1294.2 (258.3–4821.8) 1168.8 (398.2–4127.5) 0.17 1140.9 (542.1–4370.3) 1918.5 (794.1–4127.5)
516 471 524 484 485 523 522 558 518 521 486 489 523 502 521
ESR, erythrocyte sedimentation rate; LDH, lactate dehydrogenase; WBC, white blood cell.
The median follow-up time was 11.1 months (range 6–39 months). At the end of the observation period, 19 patients (32%) had died. The median survival for all patients was 26.0 months [95% confidence interval (CI) = 21–30]. The 1-, 2-, and 3-year overall survival rates were 76.3% (95% CI = 64–88), 55.6% (95% CI = 39–72), and 51.0% (95% CI = 33–69), respectively. As expected, the presence of metastasis (M1) (P < 0.001), advanced metastatic disease (M1c) (P = 0.007), anemia (P = 0.05), elevated erythrocyte sedimentation rate (P < 0.001), higher serum LDH levels (P < 0.001), and unresponsiveness to chemotherapy (P = 0.01) led to worse survival. However, neither serum fibronectin nor vitronectin concentrations played prognostic roles in survival in melanoma patients (P = 0.47 and 0.24, respectively) (Figs 1 and 2).
Discussion Adhesive interactions between tumor cells and the ECM regulate signaling events that promote cell adhesion, migration, growth, and survival. Cellular adhesion is primarily mediated by integrins, a family of cell adhesion receptors, which provide both the connection to the adhesive substrate and signaling to the actin cytoskeleton [1–19]. Alterations in the expression of adhesion molecules such as integrins have been well documented in the development of malignant melanoma [1–19]. Fibronectin is one of the most important integrin ligands of ECM, which controls many fundamental biological Fig. 1
Survival functions 1.0
0.8 Overall survival
Table 2
0.6
0.4
0.2
P = 0.04, respectively) (Table 2). However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum LDH levels, and response to chemotherapy were not found to be correlated with both serum fibronectin and vitronectin concentrations in melanoma patients (P > 0.05) (Table 3).
Fibronectin Median
0.0 0
3
6
9 12 15 18 21 24 27 30 33 36 Time (months)
Survival curves in melanoma patients according to serum fibronectin levels (P = 0.47).
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2014, Vol 24 No 5
organized network of matrix observed in the dermis. These results indicate that fibronectin was expressed in primary melanomas and corroborate the aberrant fibronectin protein expression pattern observed in melanoma cell lines [1].
Fig. 2
Survival functions 1.0
Overall survival
0.8
0.6
0.4
0.2 Vitronectin Median
0.0 0
3
6
9 12 15 18 21 24 27 30 33 36 Time (months)
Survival curves in melanoma patients according to serum vitronectin levels (P =0.24).
processes [1–8]. Abnormal expression of the fibronectin matrix protein has also been linked to metastatic melanoma progression [1–8]. There is strong evidence that the expression of fibronectin is strongly correlated with the acquisition of invasive and metastatic behavior of melanoma cells [1–8]. Importantly, blockade of fibronectin binding to melanoma cells by synthetic peptides prevented melanoma metastasis in mice [7]. These observations support the hypothesis that during melanoma development, overexpression of fibronectin can influence the control and progression of metastasis by mediating integrin-associated signaling pathways [1–8]. Fibronectin is expressed in situ during melanoma progression. Fibronectin protein is expressed at much higher levels in human melanoma cell lines compared with normal melanocytes [1,5]. Fibronectin was strongly positive in the dermis, but almost absent in the epidermis [1]. In melanoma sections, the intensity level of fibronectin staining associated with TRP-1-positive cells significantly increased compared with normal epidermis. In vertical growth-phase primary melanoma, fibronectin staining tends to be uniform throughout the tumor, whereas in radial growth-phase melanomas, the intensity varied in different areas of the tumor. This indicates that within a lesion, melanoma cells were heterogeneous for fibronectin expression and also that fibronectin immunostaining did not correlate with TRP-1 expression in these melanomas. Interestingly, in melanoma, cells appeared positive for fibronectin in the deep portion of the tumor nest and negative in the superficial portion. Finally, it may be worth noting that fibronectin localized in a punctuate pattern, which contrasted with the well-
Fibronectin was highly upregulated in these melanoma cells [1,2,5]. Clinical melanoma tissue specimens from different stages of the disease also showed a marked increase in fibronectin expression in association with enhanced invasive activity of melanoma cells [1]. The relative expression levels of fibronectin mRNAs were clearly upregulated in the primary melanomas compared with their most appropriate benign counterpart, normal melanocytic nevi. Moreover, fibronectin also induced the migration/growth of the melanoma cells [1]. The role of fibronectin expression as a prognostic indicator in sentinel lymph nodes of patients with melanoma was investigated by Gradilone et al. [8]. Fibronectin gene expression was found in 61 of 72 specimens. Moreover, a statistically significant correlation was found between negativity for fibronectin and overall survival in both univariate and multivariate analyses in addition to Clark level. These preliminary data led them to hypothesize that the absence of fibronectin in the sentinel lymph nodes microenvironment could facilitate metastatic progression of melanoma cells as well as other tumors such as gastric and colorectal malignancies. Determination of fibronectin levels in urine may be superior to quantification of serum levels. Saito et al. [21] measured the serum and urinary levels of fibronectin in 113 patients with colorectal cancer (CRC) as well as in 40 controls, with the aim of determining whether fibronectin can be used as a marker of CRC invasion or metastasis and its clinical significance. Urinary fibronectin levels were significantly higher in patients with CRC than in the controls, and both urinary and serum fibronectin levels increased with cancer progression. Patients who were positive for fibronectin tended to have a more advanced disease. High levels of fibronectin expression in both urine and serum showed a sensitivity of 80%, a specificity of 33.3%, and an accuracy of 66.6%. These results indicate that fibronectin levels increase with the progression of CRC, that fibronectin expression in urine and serum is a useful marker of the degree of disease advancement, and that fibronectin may play a role in cancer growth and development. Similarly, such a study should be planned in melanoma patients. Vitronectin is also one of the glycoproteins that play key roles in the cellular adhesion and migration of a variety of cell types [9,10]. Melanoma cells can deposit vitronectin into the ECM. It is well established that vitronectin is recognized by several cell surface receptors including integrins, uPAR, and proteoglycans [9,10]. During malignant transformation, melanoma cells start to overexpress αvβ3 integrin; this may be their mechanism for
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Fibronectin/vitronectin in melanoma Tas et al. 479
escaping apoptosis. Moreover, a large body of data on cell migration on vitronectin indicates that in most instances cell motility is dependent on the action of αvβ3 [9,11]. The risk factor for melanoma is also associated with increased levels of vitronectin in the skin [9,12], which has been observed in advanced melanoma tumors. Increased expression of αvβ3 integrin, the classic vitronectin receptor, is closely associated with increased cell invasion and metastasis [9,12–15]. Integrin αvβ3 has a limited distribution in normal tissues, is absent in normal melanocytes or benign nevi, and is overexpressed in invasive melanomas [9,16,17]. Therefore, it is suggested that αvβ3 integrin is crucial in the malignant transformation of melanoma cells [9,18,19]. Almost all findings on these ECM markers were reported by tissue–cell scale trials. Except for our study, so far, both fibronectin and vitronectin have rarely been studied in the sera of melanoma patients. A total of 60 patients with different stages of melanoma were enrolled into this study. Serum levels of these markers were analyzed quantitatively by solid-phase ELISA. In this study, we found that the baseline serum levels of both fibronectin and vitronectin of the melanoma patients were significantly higher than in those in the control group. However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum LDH levels, and response to chemotherapy were not found to be correlated with both serum assay concentrations. In addition, both serum ECM marker levels play no prognostic roles in survival in melanoma patients.
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13
Conclusion
We showed that serum levels of both fibronectin and vitronectin may be the only diagnostic marker in melanoma patients. However, both play no prognostic roles in outcome in these patients. The small sample size and the short follow-up time of our study could be considered as significant limitations and might have influenced these results. However, our study contributes to the literature because it included all stages of disease preliminarily in the literature. Further studies in a larger patient population are necessary to determine the potential clinical significance of these assays in melanoma.
14
15 16
17 18
19 20
Acknowledgements Conflicts of interest
There are no conflicts of interest.
21
Gaggioli C, Robert G, Bertolotto C, Bailet O, Abbe P, Spadafora A, et al. Tumor-derived fibronectin is involved in melanoma cell invasion and regulated by V600E B-Raf signaling pathway. J Invest Dermatol 2007; 127:400–410. Kääriäinen E, Nummela P, Soikkeli J, Yin M, Lukk M, Jahkola T, et al. Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion. J Pathol 2006; 210:181–191. Haass NK, Smalley KS, Li L, Herlyn M. Adhesion, migration and communication in melanocytes and melanoma. Pigment Cell Res 2005; 18:150–159. Kuphal S, Bauer R, Bosserhoff AK. Integrin signaling in malignant melanoma. Cancer Metastasis Rev 2005; 24:195–222. Gaggioli C, Deckert M, Robert G, Abbe P, Batoz M, Ehrengruber MU, et al. HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1. Oncogene 2005; 24:1423–1433. Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix M, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 2000; 406:536–540. Humphries MJ, Olden K, Yamada KM. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science 1986; 233:467–470. Gradilone A, Gazzaniga P, Cigna E, Vasaturo F, Vincenzi B, Gandini O, et al. Fibronectin and laminin expression in sentinel lymph nodes of patients with malignant melanoma. Br J Dermatol 2007; 157:398–401. Janik ME, Przybyło M, Pocheć E, Pokrywka M, Lityńska A. Effect of alpha3beta1 and alphavbeta3 integrin glycosylation on interaction of melanoma cells with vitronectin. Acta Biochim Pol 2010; 57:55–61. Hall DE. Vitronectin. In: Kreis T, Vale R, editors. Extracellular matrix, anchor, and adhesion proteins. New York: A Sambrook & Tooze Publication at Oxford University Press; 1999. pp. 496–498. Felding-Habermann B, Fransvea E, O’Toole TEO, Manzuk L, Faha B, Hensler M. Involvement of tumor cell integrin αvβ3 in hematogenous metastasis of human melanoma cells. Clin Exp Metastasis 2002; 19:427–436. Bafetti LM, Young TN, Itoh Y, Stack MS. Intact vitronectin induces matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2 expression and enhanced cellular invasion by melanoma cells. J Biol Chem 1998; 273:143–149. Albelda SM, Mette SA, Elder DE, Stewart R, Damjanovich L, Herlyn M, Buck CA. Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression. Cancer Res 1990; 50:6757–6764. Sanders LC, Felding-Habermann B, Mueller BM, Cheresh DA. Role of alpha V integrins and vitronectin in human melanoma cell growth. Cold Spring Harb Symp Quant Biol 1992; 57:233–240. Nip J, Brodt P. The role of the integrin vitronectin receptor, alpha v beta 3 in melanoma metastasis. Cancer Metastasis Rev 1995; 14:241–252. Baroni A, Paoletti I, Silvestri I, Buommino E, Carriero MV. Early vitronectin receptor downregulation in a melanoma cell line during all trans retinoic acidinduced apoptosis. Br J Dermatol 2003; 148:424–433. Eliceiri BP, Cheresh DA. Role of integrins during angiogenesis. Cancer J 2000; 6 (Suppl 3):S245–S249. Mitjans F, Meyer T, Fittschen C, Goodman S, Jonczyk A, Marshall JF, et al. In vivo therapy of malignant melanoma by means of antagonists of alphaV integrins. Int J Cancer 2000; 87:716–723. Seftor REB, Seftor EA, Hendrix MJ. Molecular role(s) for integrins in human melanoma invasion. Cancer Metastasis Rev 1999; 18:359–375. Sabatino M, Kim-Schulze S, Panelli MC, Stroncek D, Wang E, Taback B, et al. Serum vascular endothelial growth factor and fibronectin predict clinical response to high-dose interleukin-2 therapy. J Clin Oncol 2009; 27:2645–2652. Saito N, Nishimura H, Kameoka S. Clinical significance of fibronectin expression in colorectal cancer. Mol Med Rep 2008; 1:77–81.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Clinical significance of serum fibronectin and vitronectin levels in melanoma patients Faruk Tas, Senem Karabulut, Elif Bilgin, Didem Tastekin and Derya Duranyildiz Fibronectin and vitronectin are the important components of the extracellular matrix proteins. The aim of this study was to determine the clinical significance of these protein serum levels in patients with melanoma. A total of 60 patients with a pathologically confirmed diagnosis of melanoma were enrolled in this study. Serum fibronectin and vitronectin concentrations were determined using the solid-phase sandwich ELISA method. Thirty age-matched and sexmatched healthy controls were included in the analysis. The baseline serum fibronectin and vitronectin levels were significantly higher in patients with melanoma than those in the healthy control group (P < 0.001 and P = 0.04, respectively). However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum lactate dehydrogenase levels, and response to chemotherapy were not found to be correlated with either serum fibronectin or vitronectin concentrations (P > 0.05).
Moreover, neither serum fibronectin nor vitronectin levels played a prognostic role in outcome in melanoma patients (P = 0.47 and 0.24, respectively). In conclusion, serum levels of both fibronectin and vitronectin may be diagnostic markers in melanoma patients. However, their predictive and prognostic values were not determined. Melanoma Res 24:475–479 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Materials and methods
Melanoma is a malignancy characterized by its highly invasive and metastatic nature, properties that require interaction of the melanoma cells with the extracellular matrix (ECM) through cell adhesion, migration, proliferation, and metastasis.
Patients
Similarly, fibronectin and vitronectin, major structural components of the basement membrane, are strong promoters of cell adhesion, migration, differentiation, and proliferation through integrins and other cell surface receptors. Increased fibronectin [1–8] and vitronectin [9–19] expressions were also detected in melanoma cell lines with increased melanoma cell proliferation and metastatic potential. Although all available findings were obtained from preclinical trials, so far, a clinical study investigating the clinical significance of these ECM markers of the plasma/serum in melanoma patients has been limited [20]. Thus, the significance of the serological levels of these ECM markers in melanoma patients is not yet known. Therefore, we evaluated the soluble serum levels of fibronectin and vitronectin in melanoma patients and assessed associations with the prognosis, various known clinical variables, and response to chemotherapy to examine whether these are potential new biomarkers for use in the treatment of melanoma in this study. 0960-8931 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Melanoma Research 2014, 24:475–479 Keywords: fibronectin, melanoma, serum, vitronectin Institute of Oncology, University of Istanbul, Istanbul, Turkey Correspondence to Faruk Tas, MD, Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey Tel: + 90 212 534 80 78; fax: + 90 212 534 80 78; e-mail: [email protected] Received 22 March 2014 Accepted 6 June 2014
This study included 60 patients admitted to the Institute of Oncology, University of Istanbul, with histologically confirmed melanoma. Advanced patients with bidimensionally measurable disease without a history of chemotherapy/radiotherapy in the last 6 months were included in the study. The staging was determined according to the American Joint Committee on Cancer (AJCC) staging system. The pretreatment evaluation included a detailed assessment of clinical history and physical examination with a series of biochemistry tests including lactate dehydrogenase (LDH) and complete blood cell counts. Those with Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less and appropriate blood chemistry tests received chemotherapy on an outpatient basis comprising interferonα, cisplatin, dacarbazine, or temozolomide compounds with/without radiotherapy depending on the stage of disease. Follow-up programs consisted of clinical, laboratory, and radiological assessments performed at 8 weeks intervals during chemotherapy or every 12 weeks for no anticancer treatment. Response to treatment was determined according to revised Response Evaluation Criteria In Solid Tumors (RECIST) criteria version 1.1. For comparison of serum levels of fibronectin and vitronectin, 30 age-matched and sex-matched healthy controls DOI: 10.1097/CMR.0000000000000107
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
476
Melanoma Research
2014, Vol 24 No 5
were included in the analysis. Informed consent was obtained from all patients and the study was reviewed and approved by our local ethical committee. Measurement of serum fibronectin and vitronectin levels
Serum samples were obtained on first admission before any adjuvant and metastatic treatment was administered or follow-up of patients. Blood samples were obtained from patients with malignant melanoma and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at − 20°C until analysis. The fibronectin ELISA (eBioscience, Vienna, Austria) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human fibronectin in samples. Serum samples, standards, and biotinconjugate were added to the wells, which were precoated with human fibronectin monoclonal antibody, and allowed to incubate for 2 h. Unbound material was washed away. Fibronectin combined with streptavidin–horseradish peroxidase were added to form an immune complex and then allowed to incubate for 1 h. Unbound material was washed away. Chromogen solution was added and incubated for about 10 min (protected from light) for the conversion of the colorless solution into a blue solution, the intensity of which was proportional to the amount of fibronectin in the sample. Because of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta, Georgia, USA) at 450 nm. The results were expressed as ng/ml. The Vitronectin ELISA (Wuhan EIAab Science, Wuhan, China) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human vitronectin in samples. Serum samples and standards were added to the wells, which were precoated with human vitronectin monoclonal antibody, and allowed to incubate for 2 h. Unbound material was washed away. Vitronectin combined with streptavidin–horseradish peroxidase were added to form an immune complex and then allowed to incubate for 1 h. Unbound material was washed away. Chromogen solution was added and incubated for 15–25 min (protected from light) for the conversion of the colorless solution into a blue solution, the intensity of which was proportional to the amount of vitronectin in the sample. Because of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer) at 450 nm. The results were expressed as ng/ml. Statistical analysis
Continuous variables were categorized using median values as the cutoff point. Assessment of relationships, comparisons between various clinical/laboratory parameters
and serum levels of fibronectin and vitronectin assays were carried out using the Mann–Whitney U-test and the Kruskal–Wallis test for two and three groups, respectively. Spearman’s rank order correlation was used for correlation analysis. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. The Kaplan–Meier method was used to estimate the survival of patients and differences in survivals were assessed using the log-rank statistics. A P value of less than 0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.
Results A total of 60 patients with stage I–II (n = 13), stage III (n = 14), stage IV (n = 31), and undetermined stage (n = 2) with a pathologically confirmed diagnosis of melanoma were enrolled in this study. The majority of the patients with metastatic disease were M1c (18/31, 58%). The baseline histopathological characteristics and the demographic characteristics of the patients are listed in Table 1. The median age at diagnosis was 53.5 years (range 16–88 years); 55% of the patients in the group were men (n = 33). The baseline serum fibronectin and vitronectin levels were significantly higher in patients with melanoma than those in the healthy control group (P < 0.001 and Table 1
Patient and disease characteristics
Variables Number of patients Age (years) < 50/ ≥ 50 Sex Male/female Site of lesion Axial/extremity/unknown Histology Nodular/non-nodular/unknown Stage of disease I–II/III/IV/unknown Tumor statusa Thin (T1–T2)/thick (T3–T4)/unknown Node statusa Negative/positive M1 status M1a + b/M1c Serum hemoglobin level (12 g/dl) Low/normal/unknown Serum WBC count (10 000) Normal/elevated/unknown Serum LDH level (450 U/l) Normal/elevated ESR (40/h) Normal/elevated/unknown Response to chemotherapyb Yes/no/unknown Last status Alive/dead
n 60 26/34 33/27 38/16/6 9/31/20 13/14/31/2 10/16/1 12/14 13/18 19/40/1 50/9/1 48/12 28/20/12 10/11/10 41/19
ESR, erythrocyte sedimentation rate; LDH, lactate dehydrogenase; WBC, white blood cell. In nonmetastatic patients. b In metastatic patients. a
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Fibronectin/vitronectin in melanoma Tas et al. 477
Values of serum extracellular matrix assay levels in melanoma patients and healthy controls
No significant relationship was found between the serum levels of fibronectin and vitronectin (rs = 0.190, n = 60, P = 0.884, Spearman’s correlation).
Patients (n = 60) Range
Controls (n = 30)
Assays
Median
Median
Range
P
Fibronectin (ng/ml) Vitronectin (ng/ml)
515 362–678 192 58–532 < 0.001 1259.3 258.3–4821.8 1111.1 33.9–2944.0 0.04
Results (median and range) of comparisons between the extracellular matrix assays and various clinical parameters in melanoma patients
Table 3
Parameters Age (P) (years) Old (≥50) Young (< 50) Sex (P) Male Female Site of lesion (P) Axial Extremity Histology (P) Non-nodular Nodular Stage of disease (P) I–II III IV Tumor status (P) Thin Thick Node status (in M0 disease) (P) Negative Positive Metastasis status (in all patients) (P) 0 1 M1 status (P) a–b c Serum hemoglobin level (P) Normal Low Serum WBC count (P) Normal Elevated ESR (P) Normal Elevated Serum LDH level (P) Normal Elevated Response to chemotherapy (P) Responsive Nonresponsive
Fibronectin (ng/ml)
Vitronectin (ng/ml)
0.43 (192–678) (362–676) 0.07 (362–678) (366–678) 0.64 (362–678) (366–674) 0.43 (376–676) (362–678) 0.95 (392–674) (362–674) (366–678) 0.42 (392–674) (362–674) 0.63 (392–674) (362–674) 0.84
0.24 1160.6 (258.3–4821.8) 1354.4 (515.6–4370.3) 0.12 1060.2 (258.3–4821.8) 1461.1 (677.6–3811.3) 0.34 1259.3 (258.3–4821.8) 1784.4 (677.6–4634.4) 0.48 1316.5 (258.3–4821.8) 974.1 (542.1–4370.3) 0.93 1060.2 (258.3–4821.8) 1320.7 (581.1–4634.4) 1316.5 (398.2–4370.3) 0.14 1646.8 (774.8–4821.8) 1017.2 (258.3–4634.4) 0.56 1056.8 (258.3–4821.8) 1320.7 (581.1–4634.4) 0.77
518 (362–676) 486 (366–678) 0.54 456 (394–678) 517 (366–676) 0.38 520 (362–678) 484 (366–678) 0.14 486 (362–678) 542 (416–676) 0.81 501 (362–678) 480 (388–678) 0.77 518 (362–678) 480 (366–676) 0.39 502 (404–678) 458 (388–676)
1180.2 (258.3–4821.8) 1316.5 (398.2–4370.3) 0.83 1316.5 (677.6–3682.1) 1316.6 (398.2–4370.3) 0.89 1259.3 (258.3–4821.8) 1320.3 (398.2–4370.3) 0.72 1283.5 (258.3–4821.8) 1271.9 (398.2–4127.5) 0.33 1461.1 (581.1–4821.8) 1175.8 (258.3–4370.3) 0.57 1294.2 (258.3–4821.8) 1168.8 (398.2–4127.5) 0.17 1140.9 (542.1–4370.3) 1918.5 (794.1–4127.5)
516 471 524 484 485 523 522 558 518 521 486 489 523 502 521
ESR, erythrocyte sedimentation rate; LDH, lactate dehydrogenase; WBC, white blood cell.
The median follow-up time was 11.1 months (range 6–39 months). At the end of the observation period, 19 patients (32%) had died. The median survival for all patients was 26.0 months [95% confidence interval (CI) = 21–30]. The 1-, 2-, and 3-year overall survival rates were 76.3% (95% CI = 64–88), 55.6% (95% CI = 39–72), and 51.0% (95% CI = 33–69), respectively. As expected, the presence of metastasis (M1) (P < 0.001), advanced metastatic disease (M1c) (P = 0.007), anemia (P = 0.05), elevated erythrocyte sedimentation rate (P < 0.001), higher serum LDH levels (P < 0.001), and unresponsiveness to chemotherapy (P = 0.01) led to worse survival. However, neither serum fibronectin nor vitronectin concentrations played prognostic roles in survival in melanoma patients (P = 0.47 and 0.24, respectively) (Figs 1 and 2).
Discussion Adhesive interactions between tumor cells and the ECM regulate signaling events that promote cell adhesion, migration, growth, and survival. Cellular adhesion is primarily mediated by integrins, a family of cell adhesion receptors, which provide both the connection to the adhesive substrate and signaling to the actin cytoskeleton [1–19]. Alterations in the expression of adhesion molecules such as integrins have been well documented in the development of malignant melanoma [1–19]. Fibronectin is one of the most important integrin ligands of ECM, which controls many fundamental biological Fig. 1
Survival functions 1.0
0.8 Overall survival
Table 2
0.6
0.4
0.2
P = 0.04, respectively) (Table 2). However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum LDH levels, and response to chemotherapy were not found to be correlated with both serum fibronectin and vitronectin concentrations in melanoma patients (P > 0.05) (Table 3).
Fibronectin Median
0.0 0
3
6
9 12 15 18 21 24 27 30 33 36 Time (months)
Survival curves in melanoma patients according to serum fibronectin levels (P = 0.47).
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2014, Vol 24 No 5
organized network of matrix observed in the dermis. These results indicate that fibronectin was expressed in primary melanomas and corroborate the aberrant fibronectin protein expression pattern observed in melanoma cell lines [1].
Fig. 2
Survival functions 1.0
Overall survival
0.8
0.6
0.4
0.2 Vitronectin Median
0.0 0
3
6
9 12 15 18 21 24 27 30 33 36 Time (months)
Survival curves in melanoma patients according to serum vitronectin levels (P =0.24).
processes [1–8]. Abnormal expression of the fibronectin matrix protein has also been linked to metastatic melanoma progression [1–8]. There is strong evidence that the expression of fibronectin is strongly correlated with the acquisition of invasive and metastatic behavior of melanoma cells [1–8]. Importantly, blockade of fibronectin binding to melanoma cells by synthetic peptides prevented melanoma metastasis in mice [7]. These observations support the hypothesis that during melanoma development, overexpression of fibronectin can influence the control and progression of metastasis by mediating integrin-associated signaling pathways [1–8]. Fibronectin is expressed in situ during melanoma progression. Fibronectin protein is expressed at much higher levels in human melanoma cell lines compared with normal melanocytes [1,5]. Fibronectin was strongly positive in the dermis, but almost absent in the epidermis [1]. In melanoma sections, the intensity level of fibronectin staining associated with TRP-1-positive cells significantly increased compared with normal epidermis. In vertical growth-phase primary melanoma, fibronectin staining tends to be uniform throughout the tumor, whereas in radial growth-phase melanomas, the intensity varied in different areas of the tumor. This indicates that within a lesion, melanoma cells were heterogeneous for fibronectin expression and also that fibronectin immunostaining did not correlate with TRP-1 expression in these melanomas. Interestingly, in melanoma, cells appeared positive for fibronectin in the deep portion of the tumor nest and negative in the superficial portion. Finally, it may be worth noting that fibronectin localized in a punctuate pattern, which contrasted with the well-
Fibronectin was highly upregulated in these melanoma cells [1,2,5]. Clinical melanoma tissue specimens from different stages of the disease also showed a marked increase in fibronectin expression in association with enhanced invasive activity of melanoma cells [1]. The relative expression levels of fibronectin mRNAs were clearly upregulated in the primary melanomas compared with their most appropriate benign counterpart, normal melanocytic nevi. Moreover, fibronectin also induced the migration/growth of the melanoma cells [1]. The role of fibronectin expression as a prognostic indicator in sentinel lymph nodes of patients with melanoma was investigated by Gradilone et al. [8]. Fibronectin gene expression was found in 61 of 72 specimens. Moreover, a statistically significant correlation was found between negativity for fibronectin and overall survival in both univariate and multivariate analyses in addition to Clark level. These preliminary data led them to hypothesize that the absence of fibronectin in the sentinel lymph nodes microenvironment could facilitate metastatic progression of melanoma cells as well as other tumors such as gastric and colorectal malignancies. Determination of fibronectin levels in urine may be superior to quantification of serum levels. Saito et al. [21] measured the serum and urinary levels of fibronectin in 113 patients with colorectal cancer (CRC) as well as in 40 controls, with the aim of determining whether fibronectin can be used as a marker of CRC invasion or metastasis and its clinical significance. Urinary fibronectin levels were significantly higher in patients with CRC than in the controls, and both urinary and serum fibronectin levels increased with cancer progression. Patients who were positive for fibronectin tended to have a more advanced disease. High levels of fibronectin expression in both urine and serum showed a sensitivity of 80%, a specificity of 33.3%, and an accuracy of 66.6%. These results indicate that fibronectin levels increase with the progression of CRC, that fibronectin expression in urine and serum is a useful marker of the degree of disease advancement, and that fibronectin may play a role in cancer growth and development. Similarly, such a study should be planned in melanoma patients. Vitronectin is also one of the glycoproteins that play key roles in the cellular adhesion and migration of a variety of cell types [9,10]. Melanoma cells can deposit vitronectin into the ECM. It is well established that vitronectin is recognized by several cell surface receptors including integrins, uPAR, and proteoglycans [9,10]. During malignant transformation, melanoma cells start to overexpress αvβ3 integrin; this may be their mechanism for
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Fibronectin/vitronectin in melanoma Tas et al. 479
escaping apoptosis. Moreover, a large body of data on cell migration on vitronectin indicates that in most instances cell motility is dependent on the action of αvβ3 [9,11]. The risk factor for melanoma is also associated with increased levels of vitronectin in the skin [9,12], which has been observed in advanced melanoma tumors. Increased expression of αvβ3 integrin, the classic vitronectin receptor, is closely associated with increased cell invasion and metastasis [9,12–15]. Integrin αvβ3 has a limited distribution in normal tissues, is absent in normal melanocytes or benign nevi, and is overexpressed in invasive melanomas [9,16,17]. Therefore, it is suggested that αvβ3 integrin is crucial in the malignant transformation of melanoma cells [9,18,19]. Almost all findings on these ECM markers were reported by tissue–cell scale trials. Except for our study, so far, both fibronectin and vitronectin have rarely been studied in the sera of melanoma patients. A total of 60 patients with different stages of melanoma were enrolled into this study. Serum levels of these markers were analyzed quantitatively by solid-phase ELISA. In this study, we found that the baseline serum levels of both fibronectin and vitronectin of the melanoma patients were significantly higher than in those in the control group. However, known clinical variables including age of the patient, sex, site of lesion, histology, stage of disease, serum LDH levels, and response to chemotherapy were not found to be correlated with both serum assay concentrations. In addition, both serum ECM marker levels play no prognostic roles in survival in melanoma patients.
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Conclusion
We showed that serum levels of both fibronectin and vitronectin may be the only diagnostic marker in melanoma patients. However, both play no prognostic roles in outcome in these patients. The small sample size and the short follow-up time of our study could be considered as significant limitations and might have influenced these results. However, our study contributes to the literature because it included all stages of disease preliminarily in the literature. Further studies in a larger patient population are necessary to determine the potential clinical significance of these assays in melanoma.
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Acknowledgements Conflicts of interest
There are no conflicts of interest.
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