The RETREAT (Risk Estimation of Tumor Recurrence After Transplant) Score is a validated tool to predict post-transplant HCC recurrence risk. Alpha-fetoprotein (AFP) bound to Lens culinaris agglutinin (AFP-L3) and des-gamma-carboxyprothrombin (DCP) measured at transplant predict worse post-LT survival and may improve the RETREAT score. Our cohort comprised 284 patients transplanted for HCC who were within or downstaged to Milan, with 23 (8.1%) experiencing HCC recurrence. The modified RETREAT (mRETREAT) score assigns AFP-L3 >= 15% 2 points and DCP >= 7.5 ng/mL 3 points. Patients with a modified RETREAT score >= 4 showed a 3-year recurrence-free survival of 73.2% versus 97.8% recurrence-free survival if <4. In comparison, the original RETREAT score had a 3-year recurrence-free survival of 80.0% if >= 2 versus 98.0% if <2. mRETREAT demonstrated a superior AUC of 0.86, compared to the original RETREAT's 0.82, and enhanced calibration and accuracy with a lower Brier score (0.04). The integration of AFP-L3 and DCP into the RETREAT score appears to enhance the prediction of post-LT HCC recurrence. Given these promising results, further study in a larger multicenter cohort is warranted for empiric derivation and validation of a modified RETREAT score, including AFP-L3 and DCP.

OPINION STATEMENT:Liver transplantation for hepatocellular carcinoma (HCC) remains an evolving field. Major challenges HCC transplant patients face today include liver organ donor shortages and the need for both better pre-transplant bridging/downstaging therapies and post-transplant HCC recurrence treatment options. The advent of immunotherapy and the demonstrated efficacy of immune checkpoint inhibitors in multiple solid tumors including advanced/unresectable HCC hold promise in expanding both the neoadjuvant and adjuvant HCC transplant treatment regimen, though caution is needed with these immune modulating agents leading up to and following transplant. New options for pre-transplant HCC management will expand access to this curative option as well as ensure patients have adequate control of their HCC prior to transplant to maximize the utility of a liver donor. Machine perfusion has been an active area of investigation in recent years and could expand the organ donor pool, helping address current liver donor shortages. Finally, additional HCC biomarkers such as AFP-L3 and DCP have shown promise in improving risk stratification of HCC patients. Together, these three recent advancements will likely alter HCC transplant guidelines in the coming years.

Background & Aims: Alpha-fetoprotein (AFP) predicts hepatocellular carcinoma (HCC) recurrence after liver transplant (LT) but remains an imperfect biomarker. The role of DCP (des-gamma-carboxyprothrombin) and AFP-L3 (AFP bound to Lens culinans agglutinin) in predicting HCC recurrence remains incompletely characterized. AFP-L3 and DCP could identify patients at high risk of post-transplant HCC recurrence and serve as liver transplant exclusion criteria to defer transplant until patients receive additional risk-reducing pre-transplant locoregional therapy. Methods: This prospective cohort study included consecutive patients with HCC who underwent LT (within or down-staged to Milan criteria) between 2017 and 2022. Pre-transplant AFP, AFP-L3, and DCP measurements were obtained. The primary endpoint was the ability of biomarkers to predict HCC recurrence-free survival. Results: This cohort included 285 patients with a median age of 67 (IQR 63-71). At LT, median biomarker values were AFP 5.0 ng/ ml (IQR 3.0-12.1), AFP-L3 6.7% (0.5-13.2), and DCP 1.0 ng/ml (0.3-2.8). Most (94.7%) patients received pre-LT locoregional therapy. After a median post-LT follow-up of 3.1 years, HCC recurrence was observed in 18 (6.3%) patients. AFP-L3 and DCP outperformed AFP with C-statistics of 0.81 and 0.86 respectively, compared with 0.74 for AFP. A dual-biomarker combination of AFP-L3 >-15% and DCP >-7.5 predicted 61.1% of HCC recurrences, whereas HCC only recurred in 7 of 265 (2.6%) patients not meeting this threshold. The Kaplan-Meier recurrence-free survival rate at 3 years post-LT was 43.7% for patients with dual -positive biomarkers compared to 97.0% for all others (p <0.001). Conclusions: Dual-positivity for AFP-L3 >-15% and DCP >-7.5 strongly predicted post-LT HCC recurrence. This model could refine LT selection criteria and identify high-risk patients who require additional locoregional therapy prior to LT. (c) 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

OBJECTIVES/GOALS: Smoking is a well-established risk factor for lung cancer, but never smokers account for up to 25% of lung cancer cases. There is mounting evidence that lung cancer in never smokers is biologically distinct. We aim to characterize the genomic and immunologic features of lung adenocarcinoma in never smokers versus smokers. METHODS/STUDY POPULATION: We examined clinical, genomic, and bulk-RNA sequencing data from 499 patients in the TCGA lung adenocarcinoma cohort. Tumor mutation burden was analyzed using maftools (R package). Tumor immune characterization was completed using CIBERSORTx, a digital cytometry tool that uses single cell reference profiles to determine immune cell type frequencies from bulk-RNA sequencing data. Single cell reference profiles for 19 different immune cell types were constructed from sequencing of freshly resected lung tumor tissue from UCSF patients. Partitioning Around Medoids (PAM; R package) was used to identify distinct immune phenotypes based on immune cell composition. Fisher’s exact test was used to evaluate for associations between immune phenotypes and smoking status. RESULTS/ANTICIPATED RESULTS: Of the 499 TCGA lung adenocarcinoma patients, 75 were never smokers, 269 were female, and 246 were over the age of 65. Never smokers had lower tumor mutation burden and lower predicted neoantigen burden compared to smokers (p < 0.001). There was no difference in total tumor immune cell infiltration between never smokers and smokers. PAM yielded 2 distinct clusters/immune phenotypes. The first was enriched in M1 Macrophages, cytotoxic T Cells, helper T Cells, regulatory T Cells, and Plasma Cells. The second was enriched in plasmacytoid Dendritic Cells, M2 Macrophages, and exhausted cytotoxic T Cells. Never smoking status was associated with an increased odds of having the first immune phenotype (OR 1.95, 95% CI: 1.15 - 3.35) and this association was statistically significant (p = 0.0086). DISCUSSION/SIGNIFICANCE: Our findings suggest that never smokers have an immune phenotype that is distinct from that observed in smokers. The distinct immune characteristics we observed could explain clinical trial data suggesting immune checkpoint inhibitors are less effective in never smokers and hold implications for tailoring therapy.

3D models of cancer have the potential to improve basic, translational, and clinical studies. Patient-derived xenografts, spheroids, and organoids are broad categories of 3D models of cancer, and to date, these 3D models of cancer have been established for a variety of cancer types. In lung cancer, for example, 3D models offer a promising new avenue to gain novel insights into lung tumor biology and improve outcomes for patients afflicted with the number one cancer killer worldwide. However, the adoption and utility of these 3D models of cancer vary, and demonstrating the fidelity of these models is a critical first step before seeking meaningful applications. Here, we review use cases of current 3D lung cancer models and bioinformatic approaches to assessing model fidelity. Bioinformatics approaches play a key role in both validating 3D lung cancer models and high dimensional functional analyses to support downstream applications.

Background Major blood centers perform serologic testing on potential COVID-19 convalescent plasma donors retrospectively after blood donation. A hospital-based recruitment program for COVID-19 convalescent plasma (CCP) donors may be an efficient way to prospectively identify potential donors. Study Design and Methods Patients who recovered from known or suspected COVID-19 were identified and recruited through medical record searches and public appeals. Participants were screened with a modified donor history questionnaire (DHQ), and if eligible, were consented and tested for SARS-CoV-2 antibodies (IgG and IgM). Participants who were positive for SARS-CoV-2 IgG were referred to a local blood center for convalescent plasma collection. Results Of 179 individuals screened, 128 completed serologic testing and 89 were referred for convalescent plasma donation to a local blood center (49.7% of those screened). IgG antibodies to SARS-CoV-2 were detected in 23/51 (45.1%) of participants with suspected COVID-19 and in 66/77 (85.7%) of participants with self-reported PCR-confirmed COVID-19. Testing was performed at a median of 38 days since last symptoms. Participant age positively correlated with anti-SARS-CoV-2 IgG and IgM levels. Time since last symptoms did not correlate with IgG or IgM levels. A wide range of SARS-CoV-2 IgG levels were observed. Conclusion A hospital based CCP donor recruitment program can prospectively identify potential CCP donors. Variability in SARS-CoV-2 IgG levels has implications for selection of CCP units for transfusion.

The incredible success of cancer immunotherapies is still only limited to subsets of patients, which has motivated efforts to discover novel therapeutic targets to boost tumor killing by effector T cells. However, the translational potential of these discoveries may be limited by suboptimal correlation between cell line and animal models with human disease. Further, the majority of unbiased screening approaches are performed in cancer cells lines, rather than in the effector T cells themselves. We have developed a novel and flexible screening platform using sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to perform genome-wide CRISPR screens in primary human T cells at genome-wide scale. We have used this platform to survey the response of primary human CD8+ T cells to TCR stimulation. This screen revealed essential components of TCR signaling and inhibitors/agonists of T-cell proliferation. Targeted deletion of top candidate genes from the screen led to increased proliferation, increased expression of activation markers, and improved tumor killing in in vitro co-culture models. Coupling our screening platform with single-cell RNA-seq identified functional clustering of cell states across perturbations and showed that pooled screens in primary cells can now be analyzed at a single-cell level. In addition to finding ways to boost intrinsic T-cell activation, we also sought to adapt the SLICE screening platform to identify mediators of resistance to immunosuppressive forces in the tumor microenvironment. To model one such suppressive factor, we performed the screen in the presence of a potent adenosine agonist and found known and novel regulators of adenosine signaling in T cells. We are further integrating this platform with a two-cell co-culture system to attempt to find mechanisms of resistance to immunosuppressive cells in the tumor microenvironment. This novel platform provides the framework for unbiased, large-scale discovery of immune regulators as potential therapeutic targets in primary human T cells. This abstract is also being presented as Poster A90. Citation Format: Julia Carnevale, Eric Shifrut, Victoria Tobin, Theodore Roth, Jonathan Woo, Christina Bui, Jonathan Li, Morgan Diolaiti, Alan Ashworth, Alexander Marson. Discovery of immune regulators in primary human T cells using genome-wide CRISPR screens [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR08.

ABSTRACT Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies 1–4 . We recently developed a non-viral CRISPR–Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells 5 . Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to ‘shuttle’ the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived 6 hematopoietic stem progenitor cells (HSPCs).

Genetically-engineered immune cell therapies have been in development for decades and recently have proven effective to treat some types of cancer. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1–5. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs). Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies 1 . Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes 2–5 , but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites 6, 7 . In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction 8, 9 . Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor ( TCR ) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

NKG2D is an important immunoreceptor expressed on the surface of NK cells and some T cells. NKG2D recognizes a set of ligands typically expressed on infected or transformed cells, but recent studies have also documented NKG2D ligands on subsets of host non-tumor cells in tumor-bearing animals and humans. Here we show that in transplanted tumors and genetically engineered mouse cancer models, tumor-associated macrophages are induced to express the NKG2D ligand RAE-1δ. We find that a soluble factor produced by tumor cells is responsible for macrophage RAE-1δ induction, and we identify tumor-derived colony-stimulating factor-1 (CSF-1) as necessary and sufficient for macrophage RAE-1δ induction in vitro and in vivo. Furthermore, we show that induction of RAE-1δ on macrophages by CSF-1 requires PI3K p110α kinase signaling. Thus, production of CSF-1 by tumor cells leading to activation of PI3K p110α represents a novel cellular and molecular pathway mediating NKG2D ligand expression on tumor-associated macrophages.

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.