Supplemental Figure 3. Selected BTM geneset enrichments (FDR < 0,25) of SD (patient 002) and PD patients (004 and 008) at tumor site. Gene names of enriched genesets are publicly available online in supplemental data for original paper23.

Supplemental Table 1. Quantitative Immunohistochemistry (IHC). In the patient 002 with clinical benefit (stable disease, right two columns), IHC analysis of tumor showed increased CD4 (60x), CD8 (10x), PD1 (20x) and PDL1 (3x). In patients with disease (001, 004, 005, 008), quantitative IHC showed unchanged or decreased levels of CD4, CD8, PD-1, and PDL-1 over treatment periods. Individual antigens were quantified using the CRI Inform software (Perkin Elmer) which applies user-directed antigen thresholds to generate percentages normalized to the total tumor area.

Supplemental Figure 2. Hierarchical clustering of total gene expression of PBMC and tumor RNA samples. Only genes commonly expressed in PBMC and tumor samples are shown. Of note, up-regulation of specific genes at tumor site may come from tumor rather immune cells. On x-axis, each column represents patient at specific treatment time point, as numbered below. For example, 002- C1W1 is patient 002 at time point Cycle 1 week1. 1 - 002_Screen 12 - 004_Screen 2 - 002_C1W1 13 - 004_C1W2 3 - 002_C1W3 14 - 008_Screen 4 - 002_C1W7 15 - 008_C1W1 5 - 002_C1W10 16 - 008_C1W3 6 - 002_C2W11 17 - 008_C1W7 7 - 002_C2W12 18 - 008_C1W10 8 - 002_C2W13 9 - 002_C2W17 10 - 002_C2W20 11 - 002_C2W26

Supplemental Figure 4. Selected Broad Institute C7 immune geneset enrichments (FDR < 0,25). Gene names of enriched genesets are publicly available online at MSigDB: http://software.broadinstitute.org/gsea/msigdb/index.jsp.

Supplemental Table 2. List of genes, forming gene signature sets shown at Figure 6.

Supplemental Figure 1. Enrichments of Broad collection of immunological gene signatures in PBMC of SD (patient 002) and PD patients (patients 004 and 008). (A) non-supervised hierarchical clustering of selected gene signatures (FDR < 0,25). Gene names of enriched genesets are publicly available online at MSigDB: http://software.broadinstitute.org/gsea/msigdb/index.jsp. (B) Comparison of genesets enrichment scores between SD and PD patients. Positive values indicate enrichment in SD patient, while negative values - enrichment in PD patients. Enrichments were calculated based on outputs of GSEA analysis. X-axis: [Signal(SD) - Signal(PD)] / Signal(SD), where Signal is enrichment signal strength of geneset in patient 002 with SD response. Y-axis: [NES(SD) - NES(PD)] / NES(SD), where NES(SD) is normalized enrichment score of geneset in patient 002.

Abstract Purpose: Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), a synthetic double-stranded RNA complex, is a ligand for toll-like receptor-3 and MDA-5 that can activate immune cells, such as dendritic cells, and trigger natural killer cells to kill tumor cells. Patients and Methods: In this pilot study, eligible patients included those with recurrent metastatic disease in whom prior systemic therapy (head and neck squamous cell cancer and melanoma) failed. Patients received 2 treatment cycles, each cycle consisting of 1 mg poly-ICLC 3× weekly intratumorally (IT) for 2 weeks followed by intramuscular (IM) boosters biweekly for 7 weeks, with a 1-week rest period. Immune response was evaluated by immunohistochemistry (IHC) and RNA sequencing (RNA-seq) in tumor and blood. Results: Two patients completed 2 cycles of IT treatments, and 1 achieved clinical benefit (stable disease, progression-free survival 6 months), whereas the remainder had progressive disease. Poly-ICLC was well tolerated, with principal side effects of fatigue and inflammation at injection site ( Conclusions: Poly-ICLC was well tolerated in patients with solid cancer and generated local and systemic immune responses, as evident in the patient achieving clinical benefit. These results warrant further investigation and are currently being explored in a multicenter phase II clinical trial (NCT02423863). Clin Cancer Res; 24(20); 4937–48. ©2018 AACR.

166 Background: Poly-ICLC, a RNA complex, can activate dendritic cells and trigger NK cells to kill tumor cells. It can be given intramuscularly (IM) to induce systemic inflammation and intratumorally (IT) to induce immune infiltration of tumors. Methods: In this phase II study, eligible subjects are head and neck, and skin cancer patients with refractory or metastatic disease. In each treatment cycle, one accessible tumor site was targeted for IT injection of 1 mg of Poly-ICLC 3 times a week for 2 weeks followed by IM boosters biweekly for 6 weeks and then a 2 week rest period. Tumor biopsies were performed at baseline, week 3, and week 26. Pre- and post-vaccination tumors were evaluated by quantitative immunohistochemistry (IHC) and RNA sequencing. Blood samples were collected at baseline and prior to each cycle for immune response evaluations. Results: A phase I pilot portion of this study showed 1 patient who achieved clinical benefit with stable disease (progression-free survival of 6 months). Poly-ICLC was well tolerated with principal side effects of fatigue and inflammation at injection site (< grade 2). One case of grade 3 tumor necrosis was observed. In the patient with clinical benefit, IHC analysis of tumor showed increased CD4(60x), CD8(10x), PD-1(20x), and PD-L1(3x) compared to patients with progressive disease whose tumor biopsies showed unchanged/decreased CD4, CD8, PD-1, and PD-L1 levels over treatment period. Furthermore, RNASeq analysis of the same patient’s tumor and peripheral blood mononuclear cells (PBMC) showed dramatic changes such as upregulation of interferon-stimulated genes, chemokines, and genes associated with T cell activation and antigen presentation indicative of local and systemic immune activation in response to Poly-ICLC treatment. Conclusions: Preliminary findings show that Poly-ICLC is well tolerated in advanced solid cancer patients, and generates local immune response in tumor microenvironment and systemic immune response as evident in the patient achieving clinical benefit. These results warrant further investigation, and are currently being explored in this ongoing larger multicenter adaptive phase II clinical trial. Clinical trial information: NCT01984892.