Nevala, Wendy K.

Abstract Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

11534 Background: To date, there are no validated blood-based biomarkers of predicting response to PD-1 blockade. We previously reported that Bim is a downstream signaling molecule of the PD-1 pathway, and that measurement of Bim levels in circulating T-cells may predict and monitor responses to anti–PD-1 therapy in melanoma. We have identified the existence of sPD-L1 in cancer patients and showed that the sPD-L1 is biologically active and capable of triggering apoptosis in activated T-cells. Here we evaluated T cell Bim and sPD-L1 in the peripheral blood (PB) as biomarkers of response in a cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD1 therapy. Methods: 60 pts treated with anti-PD-1 had PB collected at baseline and at radiographic tumor evaluation. Frequencies of Bim+ T cells and Bim median fluorescence intensity (MFI) were measured by flow cytometry in gated tumor-reactive CD11ahighPD1+ CD8+ T cells. We also measured levels of sPD-L1 at baseline and serially during treatment with sPD-L1 ELISA. Baseline Bim and sPD-L1 levels and percent change in Bim levels in patients (pts) who had a radiographic response (CR/PR) were compared to those who had progressive disease (PD) at 12 wks. Results: Similarly to previously reported preliminary data, pts with objective response (CR/PR, 15/60) after 4 cycles of anti-PD1 therapy had higher frequency of Bim T cells at baseline compared to pts with PD (16/60) (mean 43% vs. 30%, P = 0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P = 0.0111). High baseline sPD-L1 were associated with progression on anti-PD1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p = 0.07, n = 13) and the levels increased by the first tumor assessment in patients resistant to anti-PD-1. Conclusions: Measurements of Bim and sPD-L1 levels may help to select patients who are likely to benefit from anti-PD1 monotherapy versus combinatorial strategies, and provide a new non-invasive way to monitor response to anti-PD-1 blockade. A larger validation study is underway.

e21054 Background: Up to 50% of patients undergoing resection for advanced melanoma experience recurrence. Identification of preoperative prognostic biomarkers is needed to ascertain risk of relapse and guide postoperative management. Lactate dehydrogenase (LDH) represents a strong prognostic factor in unresectable metastatic (stage IV) melanoma, but its relevance in patients with resected stage III or IV disease remains unknown. Methods: We retrospectively analyzed data from patients with stage III and IV melanoma who had undergone complete resection of disease and received follow-up treatment at Mayo Clinic, Rochester between January 1, 2000 and January 31, 2012. Clinical data were collected from electronic records. Survival data were estimated using the Kaplan-Meier method. Associations of preoperative LDH with time to relapse and death were evaluated using Cox proportional hazards regression models and summarized with hazard ratios and 95% confidence intervals. Results: A total of 154 subjects with resectable stage III or IV melanoma were included in the study. Median age at the time of resection was 58; 54 (35.1%) were female. One-hundred sixteen (75.3%) patients were classified as stage III and 38 (24.7%) stage IV. Adjuvant systemic treatment was administered in 75 (48.7%) patients and adjuvant radiation in 32 (20.7%). Median duration of follow-up was 4.0 years. Sixteen (10.3%) patients had preoperative LDH above the upper limit of normal. Each 50-unit increase in LDH was associated with a 15% increased risk of relapse (HR 1.15; p = 0.040) and 23% increased risk of death (HR 1.23; p = 0.001). After adjusting for age, gender, stage, number of sites, adjuvant systemic treatment, and adjuvant radiation, preoperative LDH remained associated with time to death (HR 1.25; p = 0.002). Preoperative LDH greater than the upper limit of normal was associated with increased hazard of death, both with univariate (HR 2.44; p = 0.005) and multivariate (HR 2.17; p = 0.017) analyses. Conclusions: This study supports the role of elevated preoperative LDH as a predictor of inferior outcomes in patients with advanced melanoma. Further study to correlate LDH to outcomes in the era of adjuvant immunotherapy is required.

3034 Background: Unique to modern immune therapy for cancer is that early in the course of treatment, patients frequently exhibit transient tumor enlargement (pseudo-progression, pPROG) due to tumor infiltration by lymphocytes (TIL). Currently, pPROG cannot be reliably distinguished from true tumor progression (PROG). There is a need for biomarker techniques to discriminate pPROG (effect of therapy) and PROG (therapy failure). Nuclear medicine offers radiopharmaceuticals capable of imaging immune cells; images can be fused to evaluate functional and anatomic characteristics of tumors, and potentially discriminate pPROG from PROG. Methods: In our study of metastatic melanoma patients, SPECT/CT imaging with 99mTc-interleukin-2 (99mTc-IL2) was performed to visualize TIL. Images were collected before/after 12 weeks of ipilimumab (IPI) or pembrolizomab (PEMBRO) therapy. The 99mTc-IL2 tracer was synthesized by conjugating succinimidyl-6-hydrazinopyridine-3-carboxylate (HYNIC-NHS) with commercial interleukin-2 (Aldesleukin). HYNIC-IL2 was incubated with tricine, 99mTcO4- (370-740 MBq) and SnCl2. After labelling 99mTc-IL2 was purified by reverse-phase chromatography and diluted in 5% glucose with 0.1% human albumin before injection. Five patients were enrolled in this study. Two patients failed to complete the 12 week 99mTc-IL2 scan due to discontinuation of IPI after: 1) grade 3 colitis and 2) patient refusal for adverse events attributed to IPI. No adverse events attributable to the tracer infusion were reported. Results: Metastatic lesions could be visualized by the tracer. Some lesions decreased in size, while others increased. A positive correlation was found between size and 99mTc-IL2 uptake, before and after therapy, suggesting the potential discrimination of tumor PROG (no 99mTc-IL2 uptake) from pPROG (high 99mTc-IL2 uptake). Immunohistochemical staining for TIL of 99mTc-IL2 positive and negative lesions are illustrated. Conclusions: The results demonstrate feasibility of 99mTc-IL2 imaging as a clinically useful tool capable of discriminating tumor PROG from pPROG. A clinical validation study is in progress. Clinical trial information: NCT01789827.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5726.

Abstract In patients with metastatic melanoma, high blood levels of galectin‐9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin‐9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin‐9 with macrophages. We aimed to determine whether galectin‐9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor‐supportive. It was determined that incubation of CD68 + macrophages with galectin‐9 or anti‐CD206 blocked target binding and that both CD206 and galectin‐9 were detected by immunoprecipitation of cell lysates. CD206 and galectin‐9 had a binding affinity of 2.8 × 10 −7 m . Galectin‐9 causes CD206 + macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP‐1), but less macrophage‐derived chemokine (MDC). Galectin‐9 had no effect on classical monocyte subsets, but caused expansion of the non‐classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin‐9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin‐9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti‐tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Lessons Learned Percutaneous thermal ablation combined with in situ granulocyte-macrophage colony-stimulating factor cytokine therapy was technically feasible and well tolerated. No significant clinical or immunologic responses were seen. Background Melanoma tumor-derived heat-shock proteins (HSPs) and HSP-peptide complexes can elicit protective antitumor responses. The granulocyte-macrophage colony-stimulating factor (GM-CSF) chemokine can also promote uptake and processing by professional antigen presenting cells (APCs). On this basis, we designed a pilot study of percutaneous thermal ablation as a means to induce heat-shock protein vaccination plus GM-CSF to determine safety and preliminary antitumor activity of this combination. Materials and Methods This study was designed to assess overall safety of percutaneous ablation combined with GM-CSF for unresectable, metastatic melanoma including uveal and mucosal types. All patients received heat-shock therapy (42°C for 30 minutes), then received one of three treatments: (a) intralesional GM-CSF (500 mcg standard dose); (b) radiofrequency ablation (RFA) + GM-CSF; or (c) cryoablation plus GM-CSF. The primary endpoint of the study was the induction of endogenous HSP70 and melanoma-specific cytotoxic T lymphocytes (CTL). Results Nine patients (three per study arm) were enrolled. No dose-limiting toxicity was observed as specified per protocol. All patients developed progressive disease and went on to receive alternative therapy. Median overall survival (OS) was 8.2 months (95% confidence interval [CI] 2–17.2). The study was not powered to detect a difference in clinical outcome among treatment groups. Conclusion Percutaneous thermal ablation plus GM-CSF was well tolerated, technically feasible, and demonstrated an acceptable adverse event profile comparable to conventional RFA and cryoablation. While HSP70 was induced following therapy, the degree of HSP70 elevation was not associated with clinical outcome or induced CTL responses. While percutaneous thermal ablation plus GM-CSF combinations including checkpoint inhibitors could be considered in future studies, the use of GM-CSF remains experimental and for use in the context of clinical trials.

82 Background: Metastatic malignant melanoma is a nearly uniformly fatal disease. Systemic therapy has notable but limited success. Novel therapeutic strategies are urgently needed. To that end, we’ve pursued combinatorial therapeutics using conventional chemotherapy added to an inhibitor of angiogenesis (bevacizumab) with some evidence of clinical benefit. Preliminary analysis of one of our trials suggested a previously unrecognized immunomodulatory effect of bevacizumab when added to chemotherapy in patients with metastatic melanoma, and this may have a potential impact on clinical outcome. Herein we evaluate the immunomodulatory effect of bevacizumab when added to chemotherapy in patients with metastatic melanoma. Methods: We measured levels of peripheral blood mononuclear cell phenotypes in addition to 42 cytokines, chemokines and growth factors in plasma of 55 patients who received albumin-bound paclitaxel and carboplatin (AC) and of 73 patients who received AC with bevacizumab (ACB) on clinical trials N057e and N0775 respectively. The relative percent change in PBMC cell counts and cytokine levels were determined from baseline at their restaging evaluation after two cycles of therapy. The Mann Whitney U test was used to compare responses between the groups. Results: There was a significant increase in CD197+type 1 macrophages in the group that received ACB (35.3%, -11.8-142.7%) compared to AC (-11.6%, -45.9-38.3%; p=0.004). There was a significant decrease in IL-6 in the group that received ACB (-42.4%, -87.7-0.0%) compared to AC (28.4%, -37.4-480%; p=0.0018). Conclusions: The addition of bevacizumab to chemotherapy for the treatment of metastatic melanoma modulates cellular immunity and a pro-inflammatory cytokine. Patients who received bevacizumab had a significant increase in CD197+ type 1 macrophages and a significant decrease in IL-6. Clinical significance of these findings is the subject of ongoing work.