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Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1−LAG-3−TIM-3−). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as “extreme responders”) were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

Supplementary Table legends 1-2

Therapeutic cancer vaccines targeting melanoma-associated antigens are commonly immunogenic but are rarely effective in promoting objective clinical responses. To identify critical molecules for activation of effective antitumor immunity, we have profiled autologous dendritic cell (DC) vaccines used to treat 35 patients with melanoma. We showed that checkpoint molecules induced by ex vivo maturation correlated with in vivo DC vaccine activity. Melanoma patient DCs had reduced expression of cell surface inducible T-cell costimulator ligand (ICOSL) and had defective intrinsic NF-κB signaling. Chromatin immunoprecipitation assays revealed NF-κB–dependent transcriptional regulation of ICOSL expression by DCs. Blockade of ICOSL on DCs reduced priming of antigen-specific CD8+ and CD4+ T cells from naïve donors in vitro. Concentration of extracellular/soluble ICOSL released from vaccine DCs positively correlated with patient clinical outcomes, which we showed to be partially regulated by ADAM10/17 sheddase activity. These data point to the critical role of canonical NF-κB signaling, the regulation of matrix metalloproteinases, and DC-derived ICOSL in the specific priming of cognate T-cell responses in the cancer setting. This study supports the implementation of targeted strategies to augment these pathways for improved immunotherapeutic outcomes in patients with cancer.

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1−LAG-3−TIM-3−). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as “extreme responders”) were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

Supplementary Data from Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells

Supplementary Tables 1-2

Supplementary Data from Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma

Supplementary Data from Adjuvant Pembrolizumab versus IFNα2b or Ipilimumab in Resected High-Risk Melanoma

Supplementary Figure legends

Supplementary Figures 1-6

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Patients with advanced melanoma that is resistant to PD-1 blockade therapy have limited treatment options. Vidutolimod (formerly CMP-001), a virus-like particle containing a CpG-A Toll-like receptor 9 (TLR9) agonist, may reverse PD-1 blockade resistance by triggering a strong IFN response to induce and attract antitumor T cells. In the dose-escalation part of this phase Ib study, vidutolimod was administered intratumorally at escalating doses with intravenous pembrolizumab to 44 patients with advanced melanoma who had progressive disease or stable disease on prior anti–PD-1 therapy. The combination of vidutolimod and pembrolizumab had a manageable safety profile, and durable responses were observed in 25% of patients, with tumor regression in both injected and noninjected lesions, including visceral lesions. Patients who responded to vidutolimod and pembrolizumab had noninflamed tumors at baseline and induction of an IFNγ gene signature following treatment, as well as increased systemic expression of the IFN-inducible chemokine CXCL10.Significance:In this phase Ib study in patients with advanced melanoma, intratumoral TLR9 agonist vidutolimod in combination with pembrolizumab had a manageable safety profile and showed promising clinical activity, supporting the further clinical development of vidutolimod to overcome PD-1 blockade resistance through induction of an IFN response.See related commentary by Sullivan, p. 2960.This article is highlighted in the In This Issue feature, p. 2945

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr161), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G2-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFκB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown. (Mol Cancer Res 2006;4(8):575–89)

Supplementary Table S1 from Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress

Supplemental Figure 1. Characterization of the resistant cell lines. Supplemental Figure 2. Quantification of the cell invasion from figure 1D. Supplemental Figure 3. Model showing ligand dependent and independent ephrin signaling. Supplemental Figure 4. Acute drug treatment does not induce ligand independent ephrin signaling. Supplemental Figure 5. Quantification of matrigel invasion data from Figure 2E. Supplemental Figure 6. knockdown of EphA2 does not resensitize to BRAF inhibition. Supplemental Figure 7. PI3K and AKT inhibitors block AKT signaling. Supplemental Figure 8. Removal of drug increases cell doubling time. Supplemental Figure 9. Removal of drug reverses the resistance phenotype. Supplemental Figure 10. Removal of drug reverses EphA2 signaling and melanoma invasion. Supplemental Figure 11. Time course of EphA2 induction following chronic BRAF inhibition Supplemental Table 1. Levels of EphA2 staining in primary and metastatic melanoma lesions Supplemental Table 2. Increased EphA2 staining observed in melanoma patient specimens on BRAF inhibitor trial Supplemental Table 3. Incidence of new metastases in patients treated with either vemurafenib or dacarbazine.

Plasma sulforaphane concentrations by day, dose, and pre- or post-BSE-SFN administration

Supplementary figures 1-6, supplementary tables 1-4

Supplementary Table 1: Baseline characteristics for patients included in the integrated gene set enrichment analysis (Figure 2-3) by cohort; Supplementary Table 6: The most frequent somatic alterations (alts) in patients with regionally metastatic melanoma by body mass index from The Cancer Genome Atlas cohort; Supplemental Table 7: Integrated gene set enrichment analysis for pathways associated with fatty acid metabolism by body mass index; Supplemental Table 8: Gene expression of selected genes of interest involved in fatty acid metabolism.

Integrated gene set enrichment analysis by body mass index (BMI) with correction for S phase. This figure presents a dotplot of genes differentially up- or downregulated in OW/OB patients versus NL BMI patients. Red indicates upregulation in OW/OB verse NL. The far left column is all patients, and then, an analysis controlling for sex, cohort, and tissue site is shown in the 3 columns to the right.

Differential gene expression analysis for each subgroup.

Immune cell analysis by BMI. A. Immunohistochemistry (IHC) analysis for CD8-, CD45RO-, FOXP3-, CD68-, GZMB-, PD-1-, LAG-3-, and CD3-positive cells in overweight/obese (OW/OB) verse normal (NL) tumors as defined by body mass index from the MD Anderson Cancer Center (MDA) cohort. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis for PD-L1-, CD45RO-, FOXP3-, EOMES-, GZMB-, PD-1-, TBET-, and TBET:FOXP3-positive cells in OW/OB verse NL tumors as defined by body mass index from the Gide cohort. Line represents median +/- interquartile range; each dot represents a single tumor.

Supplementary Table S1. Representativeness of study participants Supplementary Table S2. Analysis of the intracranial response duration in the COMBI-MB trial. Supplementary Table S3. Analysis of overall survival in the COMBI-MB trial. Supplementary Table S4. Baseline clinical features of the COMBI-BRV trial. Supplementary Table S5. Gene expression score (SingScore) and multiplex immunohistochemical results. Supplementary Table S6. Summary statistics of the linear mixed models utilized to assess the association between protein/pathway expression/score and biopsy sites. Supplementary Table S7. Mutation annotated file of exome sequencing from melanoma biopsies.

Integrated gene set enrichment analysis of subgroups by body mass index BMI. This figure compares gene expression between overweight/obese (OW/OB) and normal (NL) BMI patients with metastatic melanoma across subgroups by sex, cohort, and tissue site. Supplementary Figure 2 presents a dotplot of genes differentially up- or downregulated in OW/OB patients versus NL BMI patients by subgroups. Red indicates upregulation in OW/OB verse NL.

Purpose:Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI).Experimental Design:Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371).Results:DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI.Conclusions:These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies.See related commentary by Smalley, p. 5

Immune cell analysis by BMI and sex. A. Immunohistochemistry (IHC) analysis of the MDA cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis of the Gide cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor.

Reverse phase protein array of the PI3K pathway expression by BMI. Box plot represents the median bar with the box bounding IQR and whiskers the minimum and maximum values.

Direct metabolite measurements from a subset of The Cancer Genome Atlas (TCGA) cohort. Comparison of additional tricarboxylic acid cycle metabolites measured by liquid chromatography/mass spectrometry between metastatic melanoma tumors from overweight/obese (OW/OB) patients by body mass index (BMI) verse normal (NL) BMI from The Cancer Genome Atlas (TCGA). Lines represent mean +/- SEM; each dot represents a single tumor.

Differential gene expression analysis for covariates controlled.

Inverse Simpson alpha diversity scores of the fecal microbiome by BMI in patients with metastatic melanoma stratified by sex. Box plot represents the median bar with the box bounding the IQR and whiskers the most extreme points within 1.5 x IQR. The red dots represent OW/OB patients and the blue dots represent NL patients by BMI. This is a subgroup analysis of Figure 5C.

Beta-diversity analysis stratified by sex based on Bray-Curtis dissimilarity represents microbiome samples with metastatic melanoma in terms of the two top principal components (explaining around 17% of variance) obtained from the principal coordinate analysis. Red dots are from overweight/obese (OW/OB) BMI patients, and blue dots are from normal (NL) BMI patients. Shading inside the dots indicates the female gender. This is a subgroup analysis of Figure 5B.

Body mass index values by patient ID for all RNASeq cohorts.

Purpose:This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM).Patients and Methods:Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS).Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses.Results:In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105–0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06–3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321–0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25–0.78; P = 0.005).Conclusions:Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

Gene expression of major signalling pathways and multiplex immunohistochemical comparison of markers between timepoints and tumor sites. A) Gene expression of related oncogenic signalling pathways between treatment timepoints and site, B) Estimated mean difference of proteins and pathways between biopsy categories (95% confidence interval for the estimated mean difference is depicted as a segment), C) Multiplex immunohistochemical staining for key intracellular signalling proteins, D) Changes in immune intratumoral immune cell densities between treatment timepoints and site, E and F) Multiplex immunohistochemical staining depicted marked decrease in immune cell densities EDT in PT4 (steroid treated). PRE, collected prior to treatment; EDT, collected early during treatment (i.e., after 10-14 days of dabrafenib). MBM, melanoma brain metastasis; ECM, extracranial metastasis. PT, patient.

Purpose: We investigated the use of graded-dose peginterferon α-2b (Peg-IFN) in patients with stage IV melanoma overexpressing basic fibroblast growth factor (FGF-2). The primary objective was suppression of plasma FGF-2 to within reference range (≤7.5 pg/mL).Experimental Design: Plasma FGF-2 was measured at baseline (step 1), and patients with concentrations of 15 pg/mL or more were eligible for study treatment (step 2). Peg-IFN was given weekly at a starting dose of 0.5 μg/kg/wk with increment every 3 weeks based on serial FGF-2 concentrations.Results: Two hundred seven patients entered step 1; 45 (22%) overexpressed FGF-2 (median = 22 pg/dL). Twenty-nine eligible patients entered step 2 and received treatment. Patients' median age was 64 years (range, 29–84 years). Most had more than two prior therapies. FGF-2 decreased in 28 (97%) patients, with suppression to reference range in 10 (35%). Median time to FGF-2 suppression was 30 days. The best clinical responses were partial response (7%) and stable disease (17%). Median progression-free survival (PFS) and overall survival (OS) were 2.0 and 9.7 months, respectively. Patients who achieved FGF-2 suppression were more likely than those who did not to have a response or stable disease (P = 0.03). VEGF concentrations decreased in 27 patients (93%) during treatment and paralleled those of FGF-2 over time. We found no compensatory increase in VEGF among those with FGF-2 suppression.Conclusions: Graded-dose Peg-IFN suppresses FGF-2 in patients with metastatic melanoma who overexpress FGF-2. Over one third of patients had complete suppression of plasma FGF-2, which correlated with clinical response to this therapy. Clin Cancer Res; 19(23); 6597–604. ©2013 AACR.

Supplementary Data from Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

Supplemental Table 1: Genes analyzed which are known to be involved in melanoma pathogenesis Supplemental Table 2: Patient demographics and disease characteristics Supplemental Table 3: Clinical outcomes Supplemental Table 4: BRAF gene amplification is associated with BRAF somatic mutations Supplemental Table 5: RAF1 gene amplification is associated with worse PS Supplemental Figure Legends

PDF file 80K, Angiogenic factor concentrations in the plasma and urine before and after Peg-IFN treatment

Treatment significantly affects the circulating immune compartment.

Table S1- Tumor response by RECIST criteria. Table S2- Treatment details and reasons for discontinuation.

Supplemental Figure 3. BRAF and MET gene amplifications are associated with BRAF V600K mutation cohort

Supplementary Tables S1 through S3 Supplementary Figures S1 through S3

Purpose: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS).Experimental Design: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan–Meier methods.Results:BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm.Conclusions: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF. Clin Cancer Res; 20(12); 3328–37. ©2014 AACR.

Purpose: Dovitinib (TKI258) is an orally available inhibitor of fibroblast growth factor (FGF), VEGF, and platelet-derived growth factor receptors. This phase I/II dose–escalation study was conducted to evaluate the safety, pharmacodynamics, and preliminary efficacy of dovitinib in the treatment of advanced melanoma.Experimental Design: Patients with advanced melanoma resistant or refractory to standard therapies or for whom no standard therapy was available were enrolled. Dovitinib was administered at doses ranging from 200 to 500 mg/d.Results: Forty-seven patients were enrolled. The most frequently reported adverse events were fatigue (77%; grade ≥3, 28%), diarrhea (77%; grade ≥3, 11%), and nausea (77%; grade ≥3, 9%). Six dose-limiting toxicities were observed in the 400-mg and 500-mg dose cohorts, which consisted of grade 3 nausea, fatigue, and diarrhea and grade 4 fatigue events. The maximum tolerated dose was 400 mg/d. The best tumor response was stable disease, which was observed in 12 patients. Increases in plasma FGF23, VEGF, and placental growth factor and decreases in soluble VEGF receptor 2 were noted during the first cycle of treatment, consistent with FGF receptor (FGFR) and VEGF receptor (VEGFR) inhibition. Dynamic contrast-enhanced MRI analysis showed a dose-dependent decrease in tumor blood flow and vascular permeability with dovitinib therapy. A decrease in FGFR phosphorylation was observed in paired tumor biopsy samples from a patient treated with dovitinib at a dose of 400 mg/d.Conclusions: At a dose of 400 mg/d, dovitinib showed an acceptable safety profile and limited clinical benefit and inhibited FGFR and VEGFR. Clin Cancer Res; 17(23); 7451–61. ©2011 AACR.

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites.Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded.Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group.Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)

Supplementary material

Purpose: This multicenter randomized trial was designed to evaluate whether melanoma helper peptides augment cytotoxic T lymphocyte (CTL) responses to a melanoma vaccine and improve clinical outcome in patients with advanced melanoma.Experimental Design: One hundred seventy-five patients with measurable stage IV melanoma were enrolled into 4 treatment groups, vaccinated with 12 MHC class I-restricted melanoma peptides to stimulate CTL (12MP, group A), plus a tetanus peptide (group B), or a mixture of 6 melanoma helper peptides (6MHP, group C) to stimulate helper T lymphocytes (HTL), or with 6 melanoma helper peptide (6MHP) alone (group D), in incomplete Freund's adjuvant plus granulocyte macrophage colony-stimulating factor. CTL responses were assessed using an in vitro-stimulated IFN-γ ELIspot assay, and HTL responses were assessed using a proliferation assay.Results: In groups A to D, respectively, CTL response rates to 12 melanoma peptides were 43%, 47%, 28%, and 5%, and HTL response rates to 6MHP were in 3%, 0%, 40%, and 41%. Best clinical response was partial response in 7 of 148 evaluable patients (4.7%) without significant difference among study arms. Median overall survival (OS) was 11.8 months. Immune response to 6 MHP was significantly associated with both clinical response (P = 0.036) and OS (P = 0.004).Conclusion: Each vaccine regimen was immunogenic, but MHPs did not augment CTL responses to 12 melanoma peptides. The association of survival and immune response to 6MHP supports further investigation of helper peptide vaccines. For patients with advanced melanoma, multipeptide vaccines should be studied in combination with other potentially synergistic active therapies. Clin Cancer Res; 19(15); 4228–38. ©2013 AACR.

CCR Translation for the Article from Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

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Purpose: Copy number alterations have been shown to be involved in melanoma pathogenesis. The randomized phase III clinical trial E2603: carboplatin, paclitaxel, ± sorafenib (CP vs. CPS) offers a large collection of tumor samples to evaluate association of somatic mutations, genomic alterations, and clinical outcomes, prior to current FDA-approved therapies.Experimental Design: Copy number and mutational analysis on 119 pretreatment samples was performed.Results: CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors with RAF1 (cRAF) gene copy gains (HR, 0.372; P = 0.025) or CCND1 gene copy gains (HR, 0.45; P = 0.035). CPS therapy was associated with improved overall survival (OS) compared with CP in patients with tumors with KRAS gene copy gains (HR, 0.25; P = 0.035). BRAF gene copy gain and MET amplification were more common in samples with V600K versus V600E mutations (P < 0.001), which was validated in The Cancer Genome Atlas (TCGA) dataset.Conclusions: We observed improved treatment response with CPS in patients with melanoma whose tumors have RAF1 (cRAF), KRAS, or CCND1 amplification, all of which can be attributed to sorafenib targeting CRAF. These genomic alterations should be incorporated in future studies for evaluation as biomarkers. Clin Cancer Res; 22(2); 374–82. ©2015 AACR.