Recent Advances in Liver Transplantation for Hepatocellular Carcinoma.

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.

AFP-L3 and DCP strongly predict early hepatocellular carcinoma recurrence after liver transplantation.

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 culinaris 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. IMPACT AND IMPLICATIONS: Alpha-fetoprotein (AFP) is used to predict hepatocellular carcinoma (HCC) recurrence after liver transplant, but it remains an imperfect biomarker. In this prospective study, the biomarkers DCP (des-gamma-carboxyprothrombin) and AFP-L3 (AFP bound to Lens culinaris agglutinin) strongly predicted early HCC recurrence and outperformed AFP. A dual-biomarker combination of AFP-L3 ≥15% and DCP ≥7.5 predicted the majority of recurrences and could be used to further refine liver transplant eligibility criteria.

AFP-L3 and DCP are superior to AFP in predicting waitlist dropout in HCC patients: Results of a prospective study.

In patients with HCC awaiting liver transplantation (LT), there is a need to identify biomarkers that are superior to AFP in predicting prognosis. AFP-L3 and des-gamma-carboxyprothrombin (DCP) play a role in HCC detection, but their ability to predict waitlist dropout is unknown. In this prospective single-center study commenced in July 2017, 267 HCC patients had all 3 biomarkers obtained at LT listing. Among them, 96.2% received local-regional therapy, and 18.8% had an initial tumor stage beyond Milan criteria requiring tumor downstaging. At listing, median AFP was 7.0 ng/mL (IQR 3.4-21.5), median AFP-L3 was 7.1% (IQR 0.5-12.5), and median DCP was 1.0 ng/mL (IQR 0.2-3.8). After a median follow-up of 19.3 months, 63 (23.6%) experienced waitlist dropout, while 145 (54.3%) received LT, and 59 (22.1%) were still awaiting LT. Using Cox proportional hazards analysis, AFP-L3≥35% and DCP≥7.5 ng/mL were associated with increased waitlist dropout, whereas AFP at all tested cutoffs, including ≥20,≥ 100, and≥250 ng/mL was not. In a multivariable model, AFP-L3≥35% (HR 2.25, p =0.04) and DCP≥7.5 ng/mL (HR 2.20, p =0.02) remained associated with waitlist dropout as did time from HCC diagnosis to listing ≥ 1 year and increasing MELD-Na score. Kaplan-Meier probability of waitlist dropout within 2 years was 21.8% in those with AFP-L3<35% and DCP<7.5 ng/mL, 59.9% with either AFP-L3 or DCP elevated, and 100% for those with both elevated ( p <0.001). In this prospective study, listing AFP-L3% and DCP were superior to AFP in predicting waitlist dropout with the combination of AFP-L3≥35% and DCP≥7.5 ng/mL associated with a 100% risk of waitlist dropout, thus clearly adding prognostic value to AFP alone.

Bioinformatic Approaches to Validation and Functional Analysis of 3D Lung Cancer Models.

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.

Hospital-Based Donor Recruitment and Predonation Serologic Testing for COVID-19 Convalescent Plasma.

OBJECTIVES: Serologic testing for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in potential donors of coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) may not be performed until after blood donation. A hospital-based recruitment program for CCP may be an efficient way to identify potential donors prospectively. METHODS: Patients who recovered from known or suspected COVID-19 were identified and recruited through medical record searches and public appeals in March and April 2020. Participants were screened with a modified donor history questionnaire and, if eligible, were asked for consent and tested for SARS-CoV-2 antibodies (IgG and IgM). Participants positive for SARS-CoV-2 IgG were referred for CCP collection. RESULTS: Of 179 patients screened, 128 completed serologic testing and 89 were referred for CCP donation. IgG antibodies to SARS-CoV-2 were detected in 23 of 51 participants with suspected COVID-19 and 66 of 77 participants with self-reported COVID-19 confirmed by polymerase chain reaction (PCR). The anti-SARS-CoV-2 IgG level met the US Food and Drug Administration criteria for "high-titer" CCP in 39% of participants confirmed by PCR, as measured by the Ortho VITROS IgG assay. A wide range of SARS-CoV-2 IgG levels were observed. CONCLUSIONS: A hospital-based CCP donor recruitment program can prospectively identify potential CCP donors. Variability in SARS-CoV-2 IgG levels has implications for the selection of CCP units for transfusion.

Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency.

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).

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.

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.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. 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 using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Tumor-derived CSF-1 induces the NKG2D ligand RAE-1δ on tumor-infiltrating macrophages.

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.

Endothelial cells express NKG2D ligands and desensitize antitumor NK responses.

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.