Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients
Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensu...
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| Published in | Journal of translational medicine Vol. 6; no. 1; p. 61 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
BioMed Central
22.10.2008
BioMed Central Ltd BMC |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1479-5876 1479-5876 |
| DOI | 10.1186/1479-5876-6-61 |
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| Abstract | Background
HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.
Methods
Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100
209(210M)
and MART-1
26–35(27L)
, IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100
209
, gp100
pool
, MART-1
27–35
, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.
Results
The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.
Conclusion
Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. |
|---|---|
| AbstractList | Background
HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.
Methods
Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100
209(210M)
and MART-1
26–35(27L)
, IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100
209
, gp100
pool
, MART-1
27–35
, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.
Results
The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.
Conclusion
Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. HLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNgamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. The validation process demonstrated that the HLA-A2 tetramer, IFNgamma real time RT-PCR, and IFNgamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNgamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Our results demonstrated the tetramer flow cytometry assay, IFNgamma real-time RT-PCR, and INFgamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. Background HLA-A2 tetramer flow cytometry, IFN gamma real time RT-PCR and IFN gamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100 sub(209(210M) )and MART-1 sub(26-35(27L)), IFN gamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100 sub(209), gp100 sub(pool), MART-1 sub(27-35), or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFN gamma real time RT-PCR, and IFN gamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV & 20% (except ELISPOT using high PBMC numbers with %CV & 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFN gamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion Our results demonstrated the tetramer flow cytometry assay, IFN gamma real-time RT-PCR, and INF gamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. HLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.BACKGROUNDHLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNgamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.METHODSStandard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNgamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.The validation process demonstrated that the HLA-A2 tetramer, IFNgamma real time RT-PCR, and IFNgamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNgamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.RESULTSThe validation process demonstrated that the HLA-A2 tetramer, IFNgamma real time RT-PCR, and IFNgamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNgamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.Our results demonstrated the tetramer flow cytometry assay, IFNgamma real-time RT-PCR, and INFgamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.CONCLUSIONOur results demonstrated the tetramer flow cytometry assay, IFNgamma real-time RT-PCR, and INFgamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. Abstract Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. BACKGROUND: HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. METHODS: Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. RESULTS: The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. CONCLUSION: Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. |
| ArticleNumber | 61 |
| Audience | Academic |
| Author | Xu, Yuanxin Seiger, Keirsten DePalma, Kathleen Atwater, Laura Richards, Susan M Theobald, Valerie Sung, Crystal Perricone, Michael A |
| AuthorAffiliation | 1 Genzyme Corporation, One Mountain Road, Framingham, Massachusetts, MA 01701, USA |
| AuthorAffiliation_xml | – name: 1 Genzyme Corporation, One Mountain Road, Framingham, Massachusetts, MA 01701, USA |
| Author_xml | – sequence: 1 givenname: Yuanxin surname: Xu fullname: Xu, Yuanxin email: yuanxin.xu@genzyme.com organization: Genzyme Corporation – sequence: 2 givenname: Valerie surname: Theobald fullname: Theobald, Valerie organization: Genzyme Corporation – sequence: 3 givenname: Crystal surname: Sung fullname: Sung, Crystal organization: Genzyme Corporation – sequence: 4 givenname: Kathleen surname: DePalma fullname: DePalma, Kathleen organization: Genzyme Corporation – sequence: 5 givenname: Laura surname: Atwater fullname: Atwater, Laura organization: Genzyme Corporation – sequence: 6 givenname: Keirsten surname: Seiger fullname: Seiger, Keirsten organization: Genzyme Corporation – sequence: 7 givenname: Michael A surname: Perricone fullname: Perricone, Michael A organization: Genzyme Corporation – sequence: 8 givenname: Susan M surname: Richards fullname: Richards, Susan M organization: Genzyme Corporation |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/18945350$$D View this record in MEDLINE/PubMed |
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| Keywords | Tetramer Positive Cell PBMC Sample Tetramer Analysis Tetramer Assay Tetramer Binding |
| Language | English |
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| Snippet | Background
HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer... HLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer... Background HLA-A2 tetramer flow cytometry, IFN gamma real time RT-PCR and IFN gamma ELISPOT assays are commonly used as surrogate immunological endpoints for... BACKGROUND: HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer... Abstract Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for... |
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| SubjectTerms | Antigens, Neoplasm - immunology Biomedical and Life Sciences Biomedicine Calibration Cancer Care and treatment Cell Line, Tumor Enzyme-Linked Immunosorbent Assay - methods Flow cytometry Flow Cytometry - methods gp100 Melanoma Antigen Health aspects HLA-A2 Antigen - chemistry HLA-A2 Antigen - immunology Human immunodeficiency virus Humans Immunotherapy Interferon-gamma - genetics Interferon-gamma - immunology MART-1 Antigen Medicine/Public Health Melanoma - immunology Membrane Glycoproteins - immunology Methods Neoplasm Proteins - immunology Polymerase chain reaction Reference Standards Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction - methods Sensitivity and Specificity |
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| Title | Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients |
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