Rapid Screening of 4000 Individuals for Germ-line Variations in the BRAF Gene

Abstract Background: The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition.

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The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.489 ◽

1993 ◽

Vol 178 (2) ◽

pp. 489-495 ◽

Cited By ~ 638

Author(s):

V Brichard ◽

A Van Pel ◽

T Wölfel ◽

C Wölfel ◽

E De Plaen ◽

...

Keyword(s):

T Lymphocytes ◽

Cell Lines ◽

Melanoma Cell ◽

Autologous Tumor ◽

Cytolytic T Lymphocytes ◽

Pilot Studies ◽

Tyrosinase Gene ◽

Melanoma Patients ◽

Melanoma Cell Lines

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

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Vemurafenib resistant melanoma cells acquire mesenchymal stem cell-like properties

Advances in molecular oncology ◽

10.17650/2313-805x-2019-6-4-47-57 ◽

2019 ◽

Vol 6 (4) ◽

pp. 47-57

Author(s):

A. A. Vartanian ◽

O. S. Burova ◽

Kh. S. Vishnyakova ◽

I. V. Samoylenko ◽

V. A. Misyurin ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Acquired Resistance ◽

Melanoma Cells ◽

Mapk Signaling ◽

Brafv600e Mutation ◽

Acquired Drug Resistance ◽

Melanoma Patients ◽

Braf Mutant ◽

Melanoma Cell Lines

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

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Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma

International Journal of Molecular Sciences ◽

10.3390/ijms21176183 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6183

Author(s):

Beatrice Polini ◽

Sara Carpi ◽

Stefano Doccini ◽

Valentina Citi ◽

Alma Martelli ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Cutaneous Melanoma ◽

Luciferase Assay ◽

Strong Increase ◽

Over Expression ◽

Functional Features ◽

Melanoma Patients ◽

Melanoma Cell Lines

Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and –5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

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544 Prognostic biomarkers based on melanoma cell-lines in stage III melanoma patients

Journal of Investigative Dermatology ◽

10.1016/j.jid.2017.07.741 ◽

2017 ◽

Vol 137 (10) ◽

pp. S285

Author(s):

A.C. Knol ◽

J. Nguyen ◽

M. Pandolfino ◽

E. Varey ◽

M.G. Denis ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Prognostic Biomarkers ◽

Stage Iii ◽

Melanoma Patients ◽

Stage Iii Melanoma ◽

Melanoma Cell Lines

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Comprehensive upstream and downstream regulatory analyses identify miR-675-3p as a potential prognostic biomarker in melanoma

Human Cell ◽

10.1007/s13577-020-00473-0 ◽

2021 ◽

Author(s):

Cai-Chou Zhao ◽

Hao Guo ◽

Ying Wang ◽

Jiu-Hong Li

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Melanoma Cell ◽

Peripheral Blood ◽

Melanoma Patient ◽

Target Genes ◽

Prognostic Biomarker ◽

Primary Melanoma ◽

Melanoma Patients ◽

Melanoma Cell Lines

AbstractThis study assessed miR-675-3p-related regulatory mechanisms in melanoma and the clinical relevance of such regulatory activities. We downloaded miRNA mature strand expression RNA-Seq, phenotypic, and DNA methylation data pertaining to the TCGA Melanoma cohort. Differentially expressed miRNAs (DEMs) between metastatic and primary melanoma patient tissues were then identified, and miR-675-3p expression in melanoma patient peripheral blood was confirmed using the GSE20994 GEO dataset, while its expression in melanoma cell lines was evaluated via qRT-RCR. The clinical and prognostic implications of miR-675-3p in melanoma were assessed, and miR-675-3p target genes were identified using bioinformatics tools. Functional roles of this miRNA were explored via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. We identified 3 and 22 miRNAs that were up- and downregulated, respectively, in metastatic melanoma samples relative to primary melanoma samples. Upregulation of miR-675-3p was associated with poorer overall patient survival, tumor histologic grade, and Clark's level. Consistently, miR-675-3p was also overexpressed in the peripheral blood of melanoma patients relative to healthy controls, and in melanoma cell lines relative to control cells. Gene regulatory networks indicated that 32 transcription factors control miR-675-3p expression, and that it, in turn, regulates 10 target genes. KEGG analyses indicated that these genes were associated with cell cycle, transcriptional misregulation in cancer, TGF-beta signaling, and HIF-1 signaling pathways. Gain-of-function assays revealed that miR-675-3p could promote cell proliferation via accelerating cell cycle progression. Western blotting results indicated that miR-675-3p could active TGF-beta and HIF-1 signaling. Through upstream and downstream analyses of miR-675-3p-related regulatory activity, we confirmed that this miRNA participates in key melanoma-related processes and offers value as a prognostic biomarker in melanoma patients.

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Skp2, a prognostic marker and potential therapeutic target in metastatic melanoma

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.11034 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 11034-11034

Author(s):

G. Wang ◽

D. Hanniford ◽

A. Rose ◽

A. Gaziel ◽

A. Pavlick ◽

...

Keyword(s):

Metastatic Melanoma ◽

Cell Lines ◽

Melanoma Cell ◽

Primary Melanoma ◽

Melanoma Metastasis ◽

Genomic Amplification ◽

Aberrant Expression ◽

Skp2 Protein ◽

Melanoma Patients ◽

Melanoma Cell Lines

11034 Background: Skp2, a known oncogene, is overexpressed in several types of tumors and is associated with worse recurrence rate and overall survival in primary melanoma patients. Moreover, the anti-proliferative effects of Skp2 siRNA on various tumor cell lines have prompted the preclinical testing of Skp2 small molecule inhibitors. In this study, we assessed the clinical relevance and molecular mechanism(s) underlying Skp2 overexpression in metastatic melanoma patients. Methods: Skp2 protein levels were measured in 122 metastatic melanoma specimens using immunohistochemistry (IHC), and the association between Skp2 overexpression and post-recurrence survival was examined. Moreover, 22 cell lines (2 normal primary melanocytes, 2 primary immortal melanocytes, 4 primary melanoma cell lines, and 18 metastatic melanoma cell lines) were evaluated for Skp2 genomic amplification using Single Nucleotide Polymorphism (SNP) arrays (Affymetrix 6.0) and Skp2 gene expression using mRNA arrays (Affymetrix U133A 2.0) and quantitative RT-PCR. We also screened 18 cell lines for Skp2 mutation by sequencing. Results: Skp2 overexpression, defined as >25% tumor cells, was associated with shorter 3-yr post-recurrence survival (37%) compared to Skp2 expression ≤25% (55%) (HR=1.89, 95%, CI= 1.04, 3.42, p=0.04). Skp2 overexpression was significantly associated with the site of melanoma metastasis: visceral (n= 12; 89%), lymph node (n=49; 36%), brain (n=15; 14%), and soft-tissue (n=36; 6%) (p<0.001). SNP array revealed genomic amplification at the Skp2 locus in 6 (33%) metastatic cell lines and one primary melanoma cell line. Skp2 genomic amplification was associated with increased transcript expression. No Skp2 mutations were identified. Conclusions: Skp2 protein overexpression is associated with worse prognosis in metastasis in melanoma. Our results also support that gene amplification, rather than a Skp2 gene mutation, may be the major mechanism responsible for Skp2 aberrant expression in metastatic melanoma. No significant financial relationships to disclose.

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Absence of melanoma specificity in the reactivity of melanoma patients' sera with cultured allogeneic melanoma cell lines

Cancer ◽

10.1002/1097-0142(19820515)49:10<2043::aid-cncr2820491017>3.0.co;2-s ◽

1982 ◽

Vol 49 (10) ◽

pp. 2043-2048 ◽

Cited By ~ 3

Author(s):

Peter B. Dent ◽

Shuen-Kuei Liao ◽

Peter B. McCulloch ◽

Barbara R. Stone ◽

Dharam P. Singal

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Melanoma Patients ◽

Melanoma Cell Lines

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Multimarker Quantitative Real-Time PCR Detection of Circulating Melanoma Cells in Peripheral Blood: Relation to Disease Stage in Melanoma Patients

Clinical Chemistry ◽

10.1373/clinchem.2004.045096 ◽

2005 ◽

Vol 51 (6) ◽

pp. 981-988 ◽

Cited By ~ 79

Author(s):

Kazuo Koyanagi ◽

Christine Kuo ◽

Taku Nakagawa ◽

Takuji Mori ◽

Hideaki Ueno ◽

...

Keyword(s):

Real Time ◽

Cell Lines ◽

Melanoma Cell ◽

Peripheral Blood ◽

Melanoma Cells ◽

Stage I ◽

Disease Stage ◽

Ajcc Stage ◽

Melanoma Patients ◽

Melanoma Cell Lines

Abstract Background: Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients’ blood. Methods: We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. Results: All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 107 PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P <0.0001). Conclusions: Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients.

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