Eda Holl

BACKGROUND: We previously reported results from a phase 1 study testing intratumoral recombinant poliovirus, lerapolturev, in 12 melanoma patients. All 12 patients received anti-PD-1 systemic therapy before lerapolturev, and 11 of these 12 patients also received anti-PD-1 after lerapolturev. In preclinical models lerapolturev induces intratumoral innate inflammation that engages antitumor T cells. In the current study, prelerapolturev and postlerapolturev tumor biopsies and blood were evaluated for biomarkers of response. METHODS: The following analyses were performed on tumor tissue (n=11): (1) flow cytometric assessment of immune cell density, (2) NanoString Digital Spatial profiling of protein and the transcriptome, and (3) bulk RNA sequencing. Immune cell phenotypes and responsiveness to in vitro stimulation, including in vitro lerapolturev challenge, were measured in peripheral blood (n=12). RESULTS: Three patients who received anti-PD-1 therapy within 30 days of lerapolturev have a current median progression-free survival (PFS) of 2.3 years and had higher CD8+T cell infiltrates in prelerapolturev tumor biopsies relative to that of 7 patients with median PFS of 1.6 months and lower CD8+T cell infiltrates in prelerapolturev tumor biopsies. In peripheral blood, four patients with PFS 2.3 years (including three that received anti-PD-1 therapy within 30 days before lerapolturev and had higher pretreatment tumor CD8+T cell infiltrates) had significantly higher effector memory (CD8+, CCR7-, CD45RA-) but lower CD8+PD-1+ and CD4+PD-1+ cells compared with eight patients with median PFS 1.6 months. In addition, pretreatment blood from the four patients with median PFS 2.3 years had more potent antiviral responses to in vitro lerapolturev challenge compared with eight patients with median PFS 1.6 months. CONCLUSION: An inflamed pretreatment tumor microenvironment, possibly induced by prior anti-PD-1 therapy and a proficient peripheral blood pretreatment innate immune response (antiviral/interferon signaling) to lerapolturev was associated with long term PFS after intratumoral lerapolturev in a small cohort of patients. These findings imply a link between intratumoral T cell inflammation and peripheral immune function. TRIAL REGISTRATION NUMBER: NCT03712358.

INTRODUCTION: B cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients. METHODS: Flow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs. RESULTS: B cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival. CONCLUSION: These data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Activating intra-tumor innate immunity might enhance tumor immune surveillance. Virotherapy is proposed to achieve tumor cell killing, while indirectly activating innate immunity. Here, we report that recombinant poliovirus therapy primarily mediates antitumor immunotherapy via direct infection of non-malignant tumor microenvironment (TME) cells, independent of malignant cell lysis. Relative to other innate immune agonists, virotherapy provokes selective, TBK1-IRF3 driven innate inflammation that is associated with sustained type-I/III interferon (IFN) release. Despite priming equivalent antitumor T cell quantities, MDA5-orchestrated TBK1-IRF3 signaling, but not NFκB-polarized TLR activation, culminates in polyfunctional and Th1-differentiated antitumor T cell phenotypes. Recombinant type-I IFN increases tumor-localized T cell function, but does not mediate durable antitumor immunotherapy without concomitant pattern recognition receptor (PRR) signaling. Thus, virus-induced MDA5-TBK1-IRF3 signaling in the TME provides PRR-contextualized IFN responses that elicit functional antitumor T cell immunity. TBK1-IRF3 innate signal transduction stimulates eventual function and differentiation of tumor-infiltrating T cells.

BACKGROUND: In melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood. METHODS: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area. RESULTS: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years. CONCLUSION: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

OBJECTIVE: To perform an exploratory, descriptive pilot study of the systemic and local immune environment in patients with vesicoureteral reflux (VUR) and bladder-bowel dysfunction (BBD). METHODS: Consecutive children with VUR undergoing intravesical ureteral reimplantation were enrolled. Patients were assessed for presence of BBD by reported patient history and validated questionnaire. Fresh blood and bladder tissue, collected at the time of surgery, were immediately processed for analysis. Immune cell compositions were determined via flow cytometry. Immune cell activation was also defined at the time of analysis. LegendPlex assay analysis was utilized to define levels of circulating chemokines and cytokines. RESULTS: A total of 7 patients were enrolled. Although percentages of circulating immune cells in the blood of those with VUR/BBD and VUR alone were similar, within bladder tissue, VUR/BBD demonstrated increased immune infiltrates compared to VUR alone. Bladder sample analysis showed that B cells, and Effector Memory and Naïve T cell percentages were significantly increased in VUR/BBD patients compared to VUR patients. T cell expression of PD1 was increased in bladder tissues of BBD/VUR. Additionally, analysis of circulating neutrophils displayed significantly increased upregulation of PDL-1 in patients with VUR/BBD vs those with VUR only. CONCLUSION: These pilot data suggest an immune-rich microenvironment is present within VUR. Severity of inflammation appeared to correlate with presence of BBD. This implies that targeting pelvic inflammation may be a novel therapy for children with VUR- or non-VUR-related BBD. Follow-up studies are currently underway.