Melanoma gene expression profiles to identify mechanisms of tumor resistance Helena Harlin Human Immunologic Monitoring Facility University of Chicago
Introduction High frequencies of melanoma antigen-specific CD8 + T cells induced by vaccination or adoptive transfer do not always translate into tumor regression This observation has prompted investigation into the tumor microenvironment to identify potential factors that positively or negatively regulate the effector phase of an anti-tumor immune response One approach has been to characterize gene expression profiles from metastatic melanoma tumors (core biopsies or resected specimens)
Methods RNA was extracted from tumor biopsies of patients with metastatic melanoma (n=33) as well as from melanoma cell lines (n=5) and primary melanocyte cell lines (n=3) Gene array analysis was performed (Affymetrix chip U133A) Data was analyzed using dchip software, filtering for genes present in >1% of samples and with variation of std. dev./mean between 1 and 1. 735 genes with variable gene expression were identified. Genes and samples were clustered using hierarchical cluster analysis. In some cases, gene expression was analyzed using real time RT- PCR, both to confirm gene array data and to look for expression of genes not detected by the chip.
The gene array samples cluster into 3 main groups 1 3 2 Group 1 Group 3 Group 2
Tumor group 1 expresses immunologically relevant transcripts Putative positive factors: Cytokines (IL-18) Chemokine (CCL27) Cytokine receptor (IL-1R) Putative negative factors: Arginase IL-1R antagonist
Tumor group 2 expresses T cell, B cell, and other immune cell transcripts Putative positive factors: B cell markers (IgH, Igκ, Igλ) T cell markers (TcRα, β, γ) MΦ markers (MΦ scavenger receptor 1) Complement components Granzyme A, K MHC class II Chemokines (CXCL13, CXCL1, CXCL11, CXCL9, CXCL5, CCL5) Putative negative factors: Indoleamine-pyrrole 2,3 dioxygenase (IDO) Tryptophan 2,3-dioxygenase Angiogenesis factors (angiopoietin 2, angiopoietin 1, VEGF)
Tumor group 3 is characterized by a lack of immunologically relevant transcripts and includes melanoma and melanocyte cell lines Expression of various tumor markers: Cancer/testis Ags Melanoma differentiation Ags Oncogenes
82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 5 4 3 2 1 5 4 3 2 1 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 16 14 12 1 8 6 4 2 T cell, B cell and Mφ transcripts are expressed highly in group 2 tumor samples 1 3 2 TcRα TcRβ Igκ constant CD14 4 3 2 1
Representative transcripts encoding positive immune factors in tumor groups 1 and 2 IL-18 CD8α 1 2..1.2.3.4 2 4 6 8 1 12 14 16 18
IL-1 and IFN-γ transcripts are present variably in tumor samples from both group 1 and group 2 IL-1 IFN-γ 1 2 1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 1e-4 2e-4 3e-4 4e-4 5e-4 6e-4
6 5 4 3 2 1 Arginase and IDO expression levels show an inverse correlation Group 1 Group 3 Group 2 18 16 14 12 1 8 6 4 2 82 156 154 295 CL 537 Mel LP Mel DP Mel MP CL 624 CL 888 CL 28 CL 23 CL 23B 76 5 T 3629 158 98 T 6258 9 T 3232 4 IDO Arginase
Arginase expression validated by real-time RT- PCR is primarily found in group 1 samples Gene array qrt-pcr 1 2 1 2 3 4 5 6..2.4.6.8.1.12.14.16.18
IDO expression validated by real-time RT-PCR is primarily found in group 2 samples Gene array qrt-pcr 1 2 2 4 6 8 1 12 14 16 18..2.4.6.8.1.12.14.16
Regulatory T cell transcripts in tumor groups 1 and 2 FoxP3 GITR 1 2. 5.e-5 1.e-4 1.5e-4 2.e-4 2.5e-4 3.e-4 3.5e-4..2.4.6.8.1.12.14
Additional transcripts encoding putative negative regulators PD-L1 Angiopoietin 1 ND 1 2 1e-4 2e-4 3e-4 4e-4 5e-4 2 4 6 8 1 12 14 16
Survivin transcripts are widely expressed, being highest in group 3 tumor cell line samples 3 25 1 3 2 2 15 1 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 5
Conclusions Metastatic melanoma tumor samples cluster into at least three groups based on gene expression profiling Groups 1 and 2 have distinct gene expression patterns Both contain distinct sets of immunologically relevant genes Include putative negative and positive regulators of T cell function Group 3 lacks immune genes and clusters together with melanoma and primary melanocyte cell lines Suggests defects in inflammatory cell trafficking Group 1 samples express Arginase and group 2 contains all samples with IDO. Arginase and IDO expression are inversely correlated. Therefore, all tumors appear to be accounted for by a putative negative immunoregulatory process: Lack of inflammatory cell migration Expression of inhibitory factors A larger number of samples is currently being analyzed, with planned correlation with clinical outcome Therapeutic strategies to counter inhibitory influences in the tumor microenvironment should be considered for development
Acknowledgments University of Chicago Alpana Sahu Todd Kuna Functional Genomics Facility University of Virginia Michael Hanshew Craig Slingluff Amy Peterson Mark McKee Thomas Gajewski
Angiogenic factors are primarily present among group 2 tumor samples VEGF Angiopoietin 2 2 4 6 8 2 4 6 8 1 12 14
CD8α 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 18 16 14 12 1 8 6 4 2 CD8α CD8β 8 CD8b 6 4 2 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 CD4 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 3 25 2 15 1 5 CD4 B7-1 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 8 6 4 2 B7-1
B7-2 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 6 5 4 3 2 1 B7-2 CTLA-4 82 9156 9154 9295 L 537 el LP el DP l MP L 624 L 888 L 28 L 23 23B 76 5 3629 9158 98 6258 9 3232 354 7 6 5 4 3 2 1 CTLA-4