Citations

Abstract Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy. Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease showing a rapid clinical course, with a median survival of <1 year. Whole-genome and whole-exome sequencing have identified key genetic lesions in T-PLL, including structural alterations such as inversions, translocations, and large copy number variants, as well as recurrent somatic mutations of genes encoding chromatin regulators and of those in the JAK-STAT signaling pathway. Epigenetic alteration is implicated in the susceptibility and progression of both cancer and non-cancerous diseases. However, genome-wide epigenetic data has not been generated for T-PLL, which prohibits the mechanistic studies of transcriptional misregulation in T-PLL. This study used micrococcal nuclease digestion and sequencing (MNase-seq) to profile the chromatin accessible regions, i.e., gene regulatory regions such as promoters, enhancers and insulators, in both T-PLL patients and healthy individuals. Our analysis revealed the prevalence of lost open chromatin regions in T-PLL. We also generated histone H3 lysine 4 monomethylation (H3K4me1) and lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation and sequencing (ChIP-seq) data from the same samples. We identified 5,462 and 2,833 active enhancers (>=2.5 kb away from transcription start sites) that are lost and gained in T-PLL, respectively, compared to normal. The differential H3K27ac peaks are preferentially associated with the nearest genes differentially expressed between T-PLL and normal (chi-square test p <2.2e-16), indicating a role of epigenetic alteration in transcriptional misregulation. In addition, we identified alterations of active enhancers that target oncogenes with key roles in T-PLL, such as TCL1Aand MYC. Together, our analysis has provided insights into the epigenetic mechanisms that drive oncogenic activation in T-PLL. Citation Format: Shulan Tian, Henan Zhang, Eric Klee, Huihuang Yan, Wei Ding. Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2119.

7558 Background: A low absolute lymphocyte to monocyte ratio (ALC/AMC) has been found to predict decreased survival in lymphoma patients receiving chemotherapy and stem cell transplant. We report its clinical significance and additional cellular phenotype changes in patients receiving chimeric antigen receptor T-cell (CART) therapy. Methods: Records were reviewed for patients (pts) who received axicabtagene ciloleucel between 6/2016 and 12/2020. Receiver operator curve was generated using nominal logistic regression to predict CR as best response. Survivals were calculated using Kaplan- Meier method. Blood immune phenotype were assayed by multiparametric flow. Principle component analysis (PCA) was performed using ClusterVis. Results: Low ALC/AMC (≤0.8) prior to lymphodepletion (LD) chemotherapy on day -5 was associated with lower CR rate (AUC=0.68, Table). Our cohort of 81 pts had similar baseline characteristics except that noted in Table. Low ALC/AMC ratio is associated with shorter EFS and OS (EFS: 2.6 vs. 6.4 months, P<0.0001; OS: 5.3 months vs. not reached, P=0.0006), respectively. Prognostic association remained significant in multivariate analysis including ASCT, bridging therapy and CRP. Interestingly, compared to the high ALC/AMC group, the low ALC/AMC group had decreased CD8 Tem, increased CD16+CCR2+ monocytes and increased monocytes’ producing IL12, IL-10, and IL-1β (n=26). Unsupervised PCA identified 3 clusters: 1. Low ALC/AMC, all non-CR; 2. High ALC/AMC, some non-CR; 3. High ALC/AMC, all CR. Compared to cluster 1 and 2, cluster 3 had increased CD4 Tnaive, CD8 Tcm and IL-17 producing CD4 T and NK cells. Conclusions: ALC/AMC is a clinically accessible test that is strongly associated with CAR-T response and survival. Immune characterization revealed that the biologic effect is not just associated with cell ratio. Increased inflammation has been found to negatively impact CAR-T response, with some cytokines known to be from the myeloid lineage. We show that CRP is elevated in the low ALC/AMC group with increased cytokine production by monocytes. In addition, presence of T cell subset and IL-17 producing cells, before LD, are associated with clinical response. Further investigation on optimizing host immunity may help improve clinical outcome with CAR-T.[Table: see text]

Abstract While combination radiotherapy (RT) and immunotherapy is not novel there is still much to learn about this approach. Some initial pre-clinical and clinical evidence suggests triple therapy with RT and dual immune checkpoint blockade (anti-CTLA-4 + anti-PD-1/PD-L1) is a complimentary technique. We present two cases using the same novel regime of combination RT plus nivolumab and ipilimumab to treat metastatic melanoma. In both cases the burden of disease was significant within a lower extremity. Additionally, in both cases using this treatment plan, we were able establish sustained local disease control with additional systemic benefit which allowed both patients to avoid significant debility from surgical amputation. Given the response and safety we observed, this technique could be used as a potentially safe approach for future patients who are not eligible for traditional RT palliation or clinical trials.

Abstract Background: The relationship of the area of the aqueduct on quantification of the aqueductal stroke volume (SV) and max velocity need further investigation. Our aim was to assess the influence of the area of the aqueduct on quantification of the aqueductal SV and max velocity measured with phase contrast magnetic resonance imaging (PC-MRI) within the cerebral aqueduct at the level of the intercollicular sulcus.Materials and Methods: Nine healthy volunteers (mean age 29.6 yrs) were enrolled in the study and brain MRIs were performed on a 3.0T system. Quantitative analysis of aqueductal cerebrospinal fluid (CSF) flow was performed using manual regions of interest (ROI) placement. ROIs were separately drawn for each of 12 phases of the cardiac cycle, and changes in aqueduct size during the cardiac cycle were determined. Stroke volumes were calculated uses the first and ninth aqueductal ROIs and compared to each other. Max velocities at the 12 phases were also collected, and the relationship between the area and max velocity and the impact on SV were analyzed.Results: There was variation in the size of the aqueduct during the cardiac cycle, the first area（S1）was larger than the ninth（S9）. The first max velocity（Vmax1）was less than the ninth（Vmax9）. Additionally, there was a significant different between the stroke volume calculated using the first aqueductal ROI (SV1) and the ninth（SV9）. Conclusions: There is variation in the size of the cerebral aqueduct which is used to calculate stroke volume and other CSF flow parameters during the cardiac cycle. The maximum velocity may be inversely proportional to the area of ​​the aqueduct. In order to establish reliable reference values for CSF flow parameters in future studies, a variable ROI, to account for cardiac cycle variation, should be considered and incorporated.

AbstractCisplatin nephrotoxicity has been considered as serious side effect caused by cisplatin‐based chemotherapy. Recent evidence indicates that renal tubular cell apoptosis and inflammation contribute to the progression of cisplatin‐induced acute kidney injury (AKI). Hepatocyte nuclear factor 1β (HNF1β) has been reported to regulate the development of kidney cystogenesis, diabetic nephrotoxicity, etc However, the regulatory mechanism of HNF1β in cisplatin nephrotoxicity is largely unknown. In the present study, we examined the effects of HNF1β deficiency on the development of cisplatin‐induced AKI in vitro and in vivo. HNF1β down‐regulation exacerbated cisplatin‐induced RPTC apoptosis by indirectly inducing NF‐κB p65 phosphorylation and nuclear translocation. HNF1β knockdown C57BL/6 mice were constructed by injecting intravenously with HNF1β‐interfering shRNA and PEI. The HNF1β scramble and knockdown mice were treated with 30 mg/kg cisplatin for 3 days to induce acute kidney injury. Cisplatin treatment caused increased caspase 3 cleavage and p65 phosphorylation, elevated serum urea nitrogen and creatinine, and obvious histological damage of kidney such as fractured tubules in control mice, which were enhanced in HNF1β knockdown mice. These results suggest that HNF1β may ameliorate cisplatin nephrotoxicity in vitro and in vivo, probably through regulating NF‐κB signalling pathway.

Abstract Immunotherapy targeting immune checkpoints has become a common approach for many types of cancer but currently there are no reliable biomarkers to predict and monitor treatment response. Immunohistochemical staining of PD-L1, which was initially developed as a companion diagnostic, is problematic due to tumor heterogeneity, variations in specimen preparation and multiple scoring schemes. There is, therefore, a critical need for a better biomarker to guide the use of immunotherapy to improve clinical outcomes. We previously reported that higher levels of Bim and CX3CR1/granzyme B in CD8+ T cells were associated with increased response to PD-1 blockade therapy in patients with melanoma; high levels of Bim were also prognostic of poor survival in melanoma patients. In this study, we investigated the roles of Bim and CX3CR1/granzyme B in patients receiving PD-1 blockade therapy (i.e. pembrolizumab) for small bowel adenocarcinoma as part of planned correlative analysis of the ACCRU clinical trial, NCT02949219. Patients with unresectable or metastatic biopsy-proven small bowel adenocarcinoma (excluding ampulla of Vater and appendix) who had at least one prior line of systemic chemotherapy were eligible for the trial. Pembrolizumab (200 mg) was administered over 30 minutes intravenously every 3 weeks until unacceptable toxicity, disease progression, or patient refusal. All patients underwent peripheral blood collection at baseline prior to initiation of treatment and then after 3 cycles of treatment. Levels of Bim and CX3CR1/granzyme B in circulating T cells were quantified by flow cytometry using patients' peripheral blood mononuclear cells. Data was analyzed via Cox proportional hazards model, Wilcoxon Rank-Sum test, and Kaplan Meier curves using SAS 9.4. A total of 35 eligible patients were included in the analysis. Three patients had confirmed response (all partial responses) and 10 had stable disease as their best overall response. Seven patients had disease progression around the second blood draw. The median time from baseline to the second blood draw was 9 weeks. Lower levels of Bim in CD8+CD11ahi T cells at baseline on the percentage scale were associated with treatment response (median 54.5% vs. 79.5%; p=0.0087) and disease control (partial response plus stable disease; median 71.2% vs. 81.3%; p=0.0104) but higher levels led to worse progression-free survival (hazard ratio=1.05, 95% confidence interval 1.01-1.08; p=0.0051). In addition, positive changes in CX3CR1/granzyme B in CD8+CD11ahi T cells from baseline were associated with better overall survival (median 20.2 months vs. 3.7 months, p=0.0086). The levels of PD-1 and Ki-67 in CD8+CD11ahi T cells were not associated with treatment response or survival. In conclusion, Bim and CX3CR1/granzyme B are biomarkers that associate with response and survival benefits respectively of PD-1 blockade therapy. This preliminary data calls for a larger-scale, prospective study to validate their predictive and prognostic utilities. Citation Format: Mojun Zhu, Henan Zhang, Nathan R. Foster, Haidong Dong, Tanios S. Bekaii-Saab, Brandy L. Jaszewski, Patrick M. Boland, Michael J. Overman, Katrina Pedersen, Robert R. McWilliams, Academic and Community Cancer Research United (AACRU). Bim and CX3CR1/granzyme B in circulating CD8+ T cells are predictive biomarkers for PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4467.

Plants are an important source of chemically diverse natural products that target microtubules, one of the most successful targets in cancer therapy. Colchicine, paclitaxel, and vinca alkaloids are the earliest plant-derived microtubule-targeting agents (MTAs), and paclitaxel and vinca alkaloids are currently important drugs used in the treatment of cancer. Several additional plant-derived compounds that act on microtubules with improved anticancer activity are at varying stages of development. Here, we move beyond the well-discussed paclitaxel and vinca alkaloids to present other promising plant-derived MTAs with potential for development as anticancer agents. Various biological and biochemical aspects are discussed. We hope that the review will provide guidance for further exploration and identification of more effective, novel MTAs derived from plant sources.

Management of PD-1 blockade resistance in metastatic melanoma (MM) remains challenging. Immunotherapy or chemotherapy alone provides limited benefit in this setting. Chemo-immunotherapy (CIT) has demonstrated favorable efficacy and safety profiles in lung cancer. Our pre-clinical study showed that in MM patients who have failed PD-1 blockade, the addition of chemotherapy increases CX3CR1+ therapy-responsive CD8+ T-cells with enhanced anti-tumor activity, resulting in improved clinical response. Here, we examined the clinical outcomes of CIT in MM patients after PD-1 blockade failure and the treatment-related changes in CX3CR1+ therapy-responsive CD8+ T-cells. We reviewed MM patients seen between January 2012 and June 2018 who failed anti-PD-1-based therapy and received subsequent CIT, immune checkpoint inhibitors (ICI) or chemotherapy alone. Overall survival (OS), objective response rate (ORR), event-free survival (EFS), and toxicities were assessed. Among 60 patients, 33 received CIT upon disease progression on PD-1 blockade. At a median follow-up of 3.9 years, the CIT group had a median OS of 3.5 years [95% confidence interval (CI) 1.7–NR] vs. 1.8 years (95% CI 0.9–2; P = 0.002) for those who received subsequent ICI (n = 9) or chemotherapy alone (n = 18), with ORR of 59% vs. 15% (P = 0.0003), respectively. The median EFS was 7.6 months (95% CI 6–10) following CIT vs. 3.4 months (95% CI 2.8–4.1; P = 0.0005) following ICI or chemotherapy alone. Therapy-responsive CX3CR1+CD8+ T-cells showed dynamic increase with successful CIT. CIT showed favorable clinical outcomes and acceptable safety profile in PD-1 blockade-resistant patients. CX3CR1+CD8+ therapy-responsive T-cells can be potentially used for monitoring disease response to CIT.

725 Background: The impact of surgical resection of renal tumors on peripheral immune related cells is not well characterized, and has potential implications as biomarkers for systemic immune therapy are being developed. We sought to assess the effect of surgical resection on circulating cytotoxic T-cells (CTLs) for patients with renal tumors. Methods: We prospectively enrolled 40 patients undergoing partial, radical, or cytoreductive nephrectomy (PN, RN, CN) for unilateral primary renal tumors between 2016 and 2018. Blood draws were performed pre-op, 1 day post-op, and 3 months post-op. Flow cytometry was performed on peripheral blood mononuclear cells (PBMCs). The % of PBMCs expressing CD11a/CD8 (CTLs) were assessed. The % of CTLs expressing PD-1, Bim (a downstream PD-1 pathway pro-apoptotic mediator), CX3CR1/GZMB (an effector memory T-cell phenotype), and Ki67 (a proliferation marker) were assessed. Median changes in % of CTLs were evaluated with the Wilcoxon signed rank test. Comparisons between aggressive (pT3-4, N1, M1, or aggressive histology (high-grade, coagulative necrosis, sarcomatoid dedifferentiation, or specific RCC-variant histologies)) versus indolent tumors were assessed using the Wilcoxon rank sum test. Results: Twenty, 12, and 8 patients underwent RN, PN, and CN, respectively. Thirty, 7, and 3 patients had clear-cell RCC, non-clear cell RCC, and oncocytoma, respectively. Twenty-three and 17 patients had aggressive and indolent tumors, respectively. While there was no significant change at 1 day, by 3 months there were significantly increased CTLs among PBMCs (+1.3%; p=0.004). There was significant decrease in CX3CR1+GZMB+ CTLs (-2.9%; p<0.001) and significant increase in Bim+ CTLs (+3.0%; p=0.03) at 1 day but not 3 months. Interestingly, Ki67+ CTLs increased at day 1 (+0.6%; p<0.001) but decreased by 3 months (-0.6%; p=0.001). There were no significant changes in PD-1+ CTLs at either time point. There were no significant changes in any CTL characteristics at 1 day or 3 months between aggressive and indolent tumors. Conclusions: These findings characterize changing CTL profiles over time with surgical resection of renal tumors which may help guide biomarker development.