E1609 was a phase III study that enrolled patients with high-risk melanoma of cutaneous or unknown primary origin. Eligibility criteria included histological confirmation of melanoma. Patients were randomized and were rendered disease-free surgically within 12 weeks of randomization on the trial and were required to have AJCC 7th edition stages IIIB, IIIC, M1a or M1b . Other criteria included ECOG performance status (PS) of 0 or 1 and passing screening safety laboratory testing criteria. Autoimmune disorders and conditions of immunosuppression that necessitated the use of systemic corticosteroids or other immunosuppressants were not permitted.
Trial design and treatments
E1609 was an open-label phase III trial that randomized melanoma patients to systemic adjuvant therapy with ipi10, HDI or ipi3. Patients were stratified by the AJCC 7th edition stage groups of IIIB, IIIC, M1a and M1b . Clinical trial design details and additional information related to the clinical trial endpoint points, treatment regimens, randomization specifics, and trial oversight were previously published . Patient disposition is described in the consort diagram included in Additional file 1: Fig. S1. All patients provided an IRB-approved written informed consent.
Methods and statistical analysis
E1609 demonstrated significant OS benefit with ipi3 versus HDI. We investigated treatment efficacy between ipi and HDI in the subgroups by gender (female, male), age (< 55 or ≥ 55), stage at study entry (AJCC 7th edition IIIB, IIIC, M1a/1b), ECOG performance status (PS 0, 1), primary tumor ulceration (yes, no), primary tumor identification (known, unknown), number of lymph nodes involved (0, 1, 2–3, 4 +). Forest plots were created to compare OS and RFS with ipi3 versus HDI and ipi10 versus HDI using the concurrently randomized ITT populations. For the estimated HRs, 95% confidence intervals were created for all subgroups. Univariate and Multivariate analyses were conducted with the multivariate Cox regression analysis used to adjust for confounders.
Gene expression profiling (GEP)
GEP was performed on the tumor biopsies of 718 (454 male, 264 female) melanoma patients. Only metastatic tumors were included that were resected to render patients disease free prior to clinical trial enrollment. Microdissection of Formalin-Fixed Paraffin-Embedded (FFPE) tumor specimens was performed manually using an inverted microscope (Nikon Eclipse TE200) as needed to obtain a minimum of 90% tumor cells for RNA purification. Dissection involved scraping cells from unstained sections of 5 micron thickness on slides aligned in register with serially cut hematoxylin and eosin stained specimens including tumor domains demarcated by a surgical pathologist (A. K.). RNA purification was performed using the Qiagen miRNeasy FFPE Kit and protocol (Qiagen, Valencia, CA) with isolated RNA suspended in nuclease-free water. Inclusion in subsequent in vitro amplification (IVT) assays was determined both by spectrophotometric absorption ratio [260/280 > 1.8 (NanoDrop, Wilmington, DE)] and RIN values (RNA Integrity Index) determined via microchip electrophoretic analysis (Agilent Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA). We previously established that RIN values ranging from 5.0 to 8.0 in RNA from FFPE specimens can undergo successful in vitro transcription and amplification using a multiplex primer approach. Amplification was performed using the NuGen whole transcription method comprising the Ovation FFPE WTA assay (NuGEN, San Carlos, CA) employing random and 3′ primers to eliminate amplification bias beginning with 100 ng total RNA. Confirmation of cDNA diversity was obtained using the Bioanalyzer 2100 to generate an electrophoretogram for each amplification reaction regarding sample yield, integrity and size diversity compared to a laboratory human RNA standard and a Universal Human Reference RNA (Stratagene, La Jolla, CA). 5 µg of purified cDNA were incubated with fragmentation buffer (NuGEN, San Carlos, CA) at 37 °C for 30 min, then 95 °C for 2 min. The cDNA samples were hybridized on Affymetrix GeneChip HG U133A 2.0 arrays which comprehensively represent the functionally characterized human genome with overlapping probe sets for transcripts.
Data analysis of gene expression profiles
Robust Multi-array Average (RMA) method was used to normalize raw microarray data as previously described [11, 12]. Genes with multiple probe sets were collapsed by using the probe with maximum gene expression. Gene set enrichment analysis (GSEA) was performed by comparing the female and male tumor samples . In this study, KEGG pathways gene sets were obtained from MSigDB to interrogate the enrichment of pathways in the female and male samples . In order to further deconvolute the cell types in the bulk transcriptomics, we used gene sets obtained from CIBERSORT [15, 16], and TIMEx , in comparing the female versus male samples. Gene sets with a false discovery rate (FDR) q-value < 0.15 were deemed as significant. We also tested previously published prognostic gene signatures of immunotheraphy in comparing female versus male tumors including IFNγ 6-gene signature (IDO1, CXCL10, CXCL9, HLA-DRA, IFNG, STAT1) , and T-effector and IFNγ gene signature (CD8A, GZMA, GZMB, IFNG, EOMES, CXCL9, CXCL10, TBX21) . For each sample, we computed a gene signature score by averaging the standardized z-score for the genes in the signature. For each of these gene signatures . Mann–Whitney U test was performed by comparing female and male and p < 0.05 was deemed as statistically significant.
Serum and peripheral blood mononuclear cells (PBMC) data analysis
Peripheral blood samples were tested for soluble (Luminex) and cellular (multicolor flow cytometry) prognostic biomarkers in a subset of patients (N = 321; 109 female and 212 male). Mann–Whitney U test was performed by comparing between female and male and p < 0.05 was deemed as statistically significant.
Red top vacutainer tubes (BD, no anticoagulant) were used for serum collection and all samples were processed within 24 h of collection (samples received before 5 pm were processed upon receipt, those arriving after 5 pm were processed the following morning). Serum samples were centrifuged at 2500 rpm for 10 min at 4 °C according to laboratory standard operating procedures (SOPs) and single use aliquots of each patient’s sera were then stored at − 80 °C. For PBMC, blood was drawn into heparin tubes and processed by the Immunologic Monitoring Laboratory upon receipt. PBMCs were obtained from the blood samples by ficol density-gradient centrifugation and stored frozen. The laboratory freezers were monitored continuously for any temperature fluctuations and maintained the samples at -80 °C.
Multiplex serum cytokine analysis
21 serum cytokines were selected for analysis based on function. These included Th1 type cytokines (IL-12p70, IL-17, IL-2, IP-10), proinflammatory (IL-1α, IL-1β, IL-6, TNF-α, TNF-RII, IL-2R, IL-8, CRP, IL-17, IFN-α), immunoregulatory (TGF-α, IL-10, TIMP1), growth factor (VEGF-A), and other/chemokines (CCL3/MIP-1α, CCL4/MIP-1β, CXCL9/MIG, CXCL11/I-TAC). The xMAP Luminex serum assay for these cytokines was performed according to the manufacturer’s protocol (BioSource International (Camarillo, CA) as previously described , and laboratory SOPs, and analyzed on the Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, CA). Experimental data was analyzed using five-parametric curve fitting and assay controls included kit standards and multiplex QC controls (R & D Systems). Inter-assay variabilities for individual cytokines were 1.0 to 9.8% and intra-assay variabilities were 3.6 to 12.6% (information provided by Biosource International and validation performed in our laboratory). C-reactive Protein (CRP) was run singly as it requires different dilutions.
Multicolor flow cytometry
Multicolor flow cytometry was used to compare cell subset phenotypes on thawed patient peripheral blood mononuclear cells (PBMC), with healthy donor controls, run according to laboratory SOPs. Regulatory T cells (Treg) were defined as CD4+ CD25+ FOXP3+ or CD4+ CD25hi+ CD39+ cells, to incorporate the candidate functional marker CD39 as previously described . Myeloid-derived-suppressor cells (MDSC) were defined as cells expressing Lin-neg/HLA-DR−/CD33+/CD11b+ in either a “lymphocyte” (small FSCxSSC) gate, or in a “monocyte” (larger FSCxSSC) gate, and as HLA-DR+/lo CD14+ cells in a large gate as previoulsy described . We also tested the frequencies of CD4+ and CD8+ T cells specific to shared tumor-associated antigens (gp100, MART-1, NY-ESO-1) utilizing overlapping peptide libraries (15-mer peptides overlapping by 4) and a short (4–5 h) in vitro culture to identify activated (CD69+) and cytokine producing (intracellular IFNγ+) T cells. Detailed methods were described previously .