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Research Letter |

Risk for Nevus Transformation and Melanoma Proliferation and Invasion During Natalizumab Treatment Four Years of Dermoscopic Follow-up With Immunohistological Studies and Proliferation and Invasion Assays FREE

Momen Pharaon, MD1; Mélanie Tichet, MSc2; Christine Lebrun-Frénay, MD, PhD3; Sophie Tartare-Deckert, PhD2; Thierry Passeron, MD, PhD1,4
[+] Author Affiliations
1Department of Dermatology, Archet 2 Hospital, Centre Hospitalier Universitaire (CHU) Nice, Nice, France
2Institut National de la Santé et de la Recherche Médicale (INSERM), U1065, Team 11, Centre Méditerranéen de Médecin Moléculaire (C3M), Nice, France
3Department of Neurology, Pasteur Hospital, CHU Nice, Nice, France
4INSERM, U1065, Team 12, C3M, Nice, France
JAMA Dermatol. 2014;150(8):901-903. doi:10.1001/jamadermatol.2013.9411.
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Published online

Natalizumab is a monoclonal antibody targeting α4 integrin used to treat multiple sclerosis (MS). Seven cases of melanoma during natalizumab treatment have been reported since 2006.1,2 The objective of this study was to assess the effect of natalizumab on nevi transformation and tumor cell proliferation, invasion, and migration in preclinical models of melanoma.

We conducted an observational follow-up of all patients with MS treated with natalizumab in the University Hospital of Nice from January 1, 2008, through December 21, 2012. Institutional review board approval was waived. Oral informed consent was obtained for each patient. The clinical part of this study expanded a prior study3 of 44 patients from the same cohort. The evolution of nevi under treatment was assessed by clinical dermatologic examination and digital dermoscopy performed every 6 months. Nevi were examined and compared side by side with baseline photographs, and excision of suspect lesions was performed based on clinical and dermoscopic criteria. A combined in vitro approach was performed to analyze the effect of natalizumab on the proliferative, invasive, and transmigratory behavior of melanoma cells (detailed information is provided in the eMethods of the Supplement).

We included 74 patients with MS and monitored a total of 775 pigmented lesions. A mean of 11 lesions (range, 3-74) was observed for each patient. In consideration of the risk factors of melanoma, we noted that 27 patients had fair skin. Twenty-nine patients remembered experiencing frequent sunburns in childhood. Four patients had more than 50 nevi. Two patients had a familial history of melanoma. Two patients had a personal history of melanoma and were already being followed up for an atypical nevus syndrome. One of these patients had 3 melanomas removed (1 in 2006 [in situ] and 2 in 2008 [Breslow thicknesses, 0.33 and 0.36 mm]) before receiving natalizumab since 2009. The median duration of follow-up was 19 (range, 6-48) months. Twenty-three lesions (2.97%) showed modifications over time. Only 1.54% presented substantial dermoscopic changes (eFigure 1 in the Supplement). Seven melanocytic lesions were removed for histologic examination. Results for all 7 were benign. The 2 patients with a personal history of melanoma did not show any modification of their nevi.

Secreted matricellular SPARC (secreted protein acidic and rich in cysteine) and β3 integrin levels have been shown to be increased in melanoma and to promote melanoma invasion.4,5 We next analyzed whether natalizumab therapy induced changes in the expression of SPARC and β3 integrin. In the nevi removed from natalizumab-treated patients, melanocytic cells did not show a positive reaction with anti-SPARC antibody in contrast to that observed in the melanoma sample (Figure 1A). In addition, β3 integrin was virtually absent in nevi analyzed from the MS patients (Figure 1B). We then evaluated the effect of natalizumab on the proliferative and invasive phenotypes of melanoma cells in vitro. Proliferation of A375 melanoma cells treated with natalizumab was similar to that of untreated cells (eFigure 2 in the Supplement). However, a dose-dependent reduction of tumor cell migration and invasion in response to natalizumab treatment was noted (Figure 2). Natalizumab also decreased the ability of melanoma cells to migrate across endothelial monolayers (eFigure 3 in the Supplement).

Place holder to copy figure label and caption
Figure 1.
Immunofluorescent Analysis of Nevi From Natalizumab-Treated Patients With Multiple Sclerosis

A, Tissue sections stained with antibodies recognizing S100 and SPARC (secreted protein acidic and rich in cysteine). B, Tissue sections stained with antibodies recognizing β3 integrin (control serum data not shown). Antibody staining was followed by fluorescent green or red antibody conjugated secondary 6 antibody staining (Alexa Fluor 448 and 594, respectively; Molecular Probes, Inc). Images were obtained with a confocal microscope (objective ×40). A primary melanoma lesion is shown as a positive control for SPARC expression. Staining for S100 was used to identify melanocytic skin cells. In the melanoma tumor analyzed, a high correspondence between S100-positive melanoma cells and SPARC expression was found, whereas S100-positive nevi analyzed are negative for SPARC or β3 integrin melanoma progression markers.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Response to Natalizumab Treatment

A, Natalizumab decreased melanoma cell migration in a dose-dependent manner. A375 cells were detached and incubated with isotype-matched IgG (100 μg/mL) or the indicated concentrations of natalizumab for 90 minutes. Migration assays were then performed on fibronectin-coated polycarbonate cell culture inserts (Transwells; Sigma-Aldrich). After 4 hours, migrating cells on the underside of insert filters were fixed, stained, and counted under a microscope. Columns indicate the mean of 3 independent determinations; error bars, SD. B, Natalizumab reduced invasive outgrowth of melanoma spheroid in 3-dimensional collagen (described in the eMethods of the Supplement). Preformed melanoma spheroids of WM793 cells were implanted into a gel of collagen type I. Spheroids were incubated in growth medium in the presence of 75 μg/mL of isotype-matched IgG or natalizumab in growth medium for 3 days. Tumor cell outgrowth was visualized by phase contrast microscopy. Representative examples of 2 spheroids from each culture are shown (original magnification ×5).

Graphic Jump Location

Among the 74 patients with 775 monitored melanocytic skin lesions followed up for more than 4 years, substantial dermoscopic changes were observed in only 1.54% of the cases. Results of histologic analysis revealed all the excised lesions to be benign, and no melanoma was diagnosed. After a mean duration of follow-up of 14 (range, 6-20) months of the same cohort with 44 patients included at that time and 248 nevi examined, substantial dermoscopic changes were observed in 4.8% of the cases.3 In an untreated population, substantial dermoscopic changes were observed in about 4% of common and atypical nevi after a median total follow-up of 11 (range, 3-21) months.6

Unlike melanoma, the nevi removed during the follow-up of the patients treated with natalizumab did not show detectable levels of SPARC and β3 integrin, indicating that natalizumab therapy did not modulate expression of melanoma progression markers in nevi. Melanoma cell migration and invasion were significantly and proportionally reduced with increasing doses of natalizumab. Natalizumab also inhibited transendothelial migration across activated vascular cell adhesion molecule 1–expressing endothelial cells.

Collectively, these results further support that natalizumab therapy does not increase the risk for nevus transformation and that natalizumab exerts anti-invasive and antimigratory activities in vitro. Because these capabilities of malignant cells correlate with their metastatic potency, our findings provide evidence that natalizumab might have a protective effect on melanoma development and give reassuring data for its use in treatment of MS.

Corresponding Author: Thierry Passeron, MD, PhD, Department of Dermatology, Archet 2 Hospital, Centre Hospitalier Universitaire Nice, 150 route de Ginestiere, 06200 Nice, France (passeron@unice.fr).

Accepted for Publication: October 30, 2013.

Published Online: June 11, 2014. doi:10.1001/jamadermatol.2013.9411.

Author Contributions: Drs Tartare-Deckert and Passeron had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed equally to this work.

Study concept and design: Lebrun-Frénay, Tartare-Deckert, Passeron.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Pharaon, Tichet, Lebrun-Frénay.

Critical revision of the manuscript for important intellectual content: Pharaon, Lebrun-Frénay, Tartare-Deckert, Passeron.

Statistical analysis: Pharaon, Tichet.

Obtained funding: Tartare-Deckert.

Administrative, technical, or material support: Lebrun-Frénay, Tartare-Deckert, Passeron.

Study supervision: Lebrun-Frénay, Tartare-Deckert, Passeron.

Conflict of Interest Disclosures: Dr Lebrun-Frénay participated in meetings sponsored by and received honoraria for lectures, advisory board participation, and consultations from Allergan, Almirall, Biogen Idec, Bayer Schering, Genzyme, Merck Serono, Novartis, sanofi-aventis, and Teva. Dr Passeron participated in meetings sponsored by and received honoraria for lectures, advisory board participation, and consultations from Abbott, Almirall, Bioderma, Candela/Syneron, Beiersdorf, Galderma, GSK, Jansen, L’Oreal, Léo-Pharma, Merck, Pierre Fabre, Pfizer, and Sinclair-Pharma. No other disclosures were reported.

Funding/Support: This study was supported by INSERM and the Fondation ARC.

Role of the Sponsors: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank the C3M imaging core facility (Microscopy and Imaging platform Côte d’Azur [MICA]) for immunofluorescent analysis. Emeline Castela, MD (Department of Dermatology, CHU Nice), Muriel Laffon, MD (Department of Neurology, CHU Nice), Philippe Bahadoran, MD, PhD (Department of Dermatology and Centre de Recherche Clinique, CHU Nice), Fanny Rocher, PharmD (Centre de Pharmacovigilance, CHU Nice), Mikael Cohen, MD (Department of Neurology, CHU Nice), Patricia Abbe, BSc (INSERM, U1065, Team 11, C3M), Nathalie Cardot-Leccia, MD (Department of Pathology, CHU Nice), and Jean-Philippe Lacour, MD (Department of Dermatology, CHU Nice), provided medical and technical help. None received financial compensation for their services.

Polman  CH, O’Connor  PW, Havrdova  E,  et al; AFFIRM Investigators.  A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med. 2006;354(9):899-910.
PubMed   |  Link to Article
Laroni  A, Bedognetti  M, Uccelli  A, Capello  E, Mancardi  GL.  Association of melanoma and natalizumab therapy in the Italian MS population: a second case report. Neurol Sci. 2011;32(1):181-182.
PubMed   |  Link to Article
Castela  E, Lebrun-Frénay  C, Laffon  M,  et al.  Evolution of nevi during treatment with natalizumab: a prospective follow-up of patients treated with natalizumab for multiple sclerosis. Arch Dermatol. 2011;147(1):72-76.
PubMed   |  Link to Article
Sturm  RA, Satyamoorthy  K, Meier  F,  et al.  Osteonectin/SPARC induction by ectopic β3 integrin in human radial growth phase primary melanoma cells. Cancer Res. 2002;62(1):226-232.
PubMed
Robert  G, Gaggioli  C, Bailet  O,  et al.  SPARC represses E-cadherin and induces mesenchymal transition during melanoma development. Cancer Res. 2006;66(15):7516-7523.
PubMed   |  Link to Article
Kittler  H, Seltenheim  M, Dawid  M, Pehamberger  H, Wolff  K, Binder  M.  Frequency and characteristics of enlarging common melanocytic nevi. Arch Dermatol. 2000;136(3):316-320.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
Immunofluorescent Analysis of Nevi From Natalizumab-Treated Patients With Multiple Sclerosis

A, Tissue sections stained with antibodies recognizing S100 and SPARC (secreted protein acidic and rich in cysteine). B, Tissue sections stained with antibodies recognizing β3 integrin (control serum data not shown). Antibody staining was followed by fluorescent green or red antibody conjugated secondary 6 antibody staining (Alexa Fluor 448 and 594, respectively; Molecular Probes, Inc). Images were obtained with a confocal microscope (objective ×40). A primary melanoma lesion is shown as a positive control for SPARC expression. Staining for S100 was used to identify melanocytic skin cells. In the melanoma tumor analyzed, a high correspondence between S100-positive melanoma cells and SPARC expression was found, whereas S100-positive nevi analyzed are negative for SPARC or β3 integrin melanoma progression markers.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Response to Natalizumab Treatment

A, Natalizumab decreased melanoma cell migration in a dose-dependent manner. A375 cells were detached and incubated with isotype-matched IgG (100 μg/mL) or the indicated concentrations of natalizumab for 90 minutes. Migration assays were then performed on fibronectin-coated polycarbonate cell culture inserts (Transwells; Sigma-Aldrich). After 4 hours, migrating cells on the underside of insert filters were fixed, stained, and counted under a microscope. Columns indicate the mean of 3 independent determinations; error bars, SD. B, Natalizumab reduced invasive outgrowth of melanoma spheroid in 3-dimensional collagen (described in the eMethods of the Supplement). Preformed melanoma spheroids of WM793 cells were implanted into a gel of collagen type I. Spheroids were incubated in growth medium in the presence of 75 μg/mL of isotype-matched IgG or natalizumab in growth medium for 3 days. Tumor cell outgrowth was visualized by phase contrast microscopy. Representative examples of 2 spheroids from each culture are shown (original magnification ×5).

Graphic Jump Location

Tables

References

Polman  CH, O’Connor  PW, Havrdova  E,  et al; AFFIRM Investigators.  A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med. 2006;354(9):899-910.
PubMed   |  Link to Article
Laroni  A, Bedognetti  M, Uccelli  A, Capello  E, Mancardi  GL.  Association of melanoma and natalizumab therapy in the Italian MS population: a second case report. Neurol Sci. 2011;32(1):181-182.
PubMed   |  Link to Article
Castela  E, Lebrun-Frénay  C, Laffon  M,  et al.  Evolution of nevi during treatment with natalizumab: a prospective follow-up of patients treated with natalizumab for multiple sclerosis. Arch Dermatol. 2011;147(1):72-76.
PubMed   |  Link to Article
Sturm  RA, Satyamoorthy  K, Meier  F,  et al.  Osteonectin/SPARC induction by ectopic β3 integrin in human radial growth phase primary melanoma cells. Cancer Res. 2002;62(1):226-232.
PubMed
Robert  G, Gaggioli  C, Bailet  O,  et al.  SPARC represses E-cadherin and induces mesenchymal transition during melanoma development. Cancer Res. 2006;66(15):7516-7523.
PubMed   |  Link to Article
Kittler  H, Seltenheim  M, Dawid  M, Pehamberger  H, Wolff  K, Binder  M.  Frequency and characteristics of enlarging common melanocytic nevi. Arch Dermatol. 2000;136(3):316-320.
PubMed   |  Link to Article

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Supplement.

eMethods. Study design

eReferences.

eFigure 1. Dermoscopy findings in a representative nevus

eFigure 2. Effect of natalizumab on proliferation of melanoma cells

eFigure 3. Natalizumab blocks melanoma cell migration across tumor necrosis factor (TNF)–activated endothelial cells

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