Citations

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

Introduction. The impact of renal allograft rejection treatment on infection development has not been formally defined in the literature. Methods. We conducted a retrospective cohort study of 185 rejection (case) and 185 nonrejection (control) kidney transplant patients treated at our institution from 2014 to 2020 to understand the impact of rejection on infection development. Propensity scoring was used to match cohorts. We collected data for infections within 6 months of rejection for the cases and 18 months posttransplant for controls. Results. In 370 patients, we identified 466 infections, 297 in the controls, and 169 in the cases. Urinary tract infections (38.9%) and cytomegalovirus viremia (13.7%) were most common. Cumulative incidence of infection between the case and controls was 2.17 (CI 1.54–3.05); p<0.001. There was no difference in overall survival (HR 0.90, CI 0.49–1.66) or graft survival (HR 1.27, CI 0.74–2.20) between the groups. There was a significant difference in overall survival (HR 2.28, CI 1.14–4.55; p=0.019) and graft survival (HR 1.98, CI 1.10–3.56; p=0.023) when patients with infection were compared to those without. Conclusions. As previously understood, rejection treatment is a risk factor for subsequent infection development. Our data have defined this relationship more clearly. This study is unique, however, in that we found that infections, but not rejection, negatively impacted both overall patient survival and allograft survival, likely due to our institution’s robust post-rejection protocols. Clinicians should monitor patients closely for infections in the post-rejection period and have a low threshold to treat these infections while also restarting appropriate prophylaxis.

Abstract Objective Results of ethanol ablation (EA) for controlling neck nodal metastases (NNM) in adult patients with papillary thyroid carcinoma (APTC) beyond 6 months have rarely been reported. We now describe outcome results in controlling 71 NNM in 40 node-positive stage I APTC patients followed for 66 to 269 months. Methods All 40 patients were managed with bilateral thyroidectomy and radioiodine therapy and followed with neck ultrasound (US) for &gt;48 months after EA. Cumulative radioiodine doses ranged from 30 to 550 mCi; pre-EA 27 patients (67%) had 36 additional neck surgeries. Cytologic diagnosis of PTC in 71 NNM selected for EA was confirmed by US-guided biopsy. EA technique and follow-up protocol were as previously described. Results The 40 patients had 1 to 4 NNM; 67/71 NNM (94%) received 2 to 4 ethanol injections (total median volume 0.8 cc). All ablated 71 NNM shrank (mean volume reduction of 93%); nodal hypervascularity was eliminated. Thirty-eight NNM (54%) with initial volumes of 12-1404 mm3 (median 164) disappeared on neck sonography. Thirty-three hypovascular foci from ablated NNM (pre-EA volume range 31-636 mm3; median 147) were still identifiable with volume reductions of 45% to 97% observed (median 81%). There were no complications and no postprocedure hoarseness. Final results were considered to be ideal or near ideal in 55% and satisfactory in 45%. There was no evidence of tumor regrowth after EA. Conclusion Our results demonstrate that for patients with American Joint Committee on Cancer stage I APTC, who do not wish further surgery or radioiodine, and are uncomfortable with active surveillance, EA can achieve durable control of recurrent NNM.

Background: There are a lack of clinical prediction tools in early-stage Hodgkin lymphoma (E-HL). We developed and validated a modern-day model, known as E-HIPI, to predict progression-free survival (PFS) within the first 5 years (y) incorporating detailed individual patient (pt) data from international clinical trials and prospective registry data that were standardized, normalized & harmonized as part of the HoLISTIC Consortium (www.hodgkinconsortium.org). Methods: Model development utilized Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) guidelines (Moons, Ann Intern Med 2015) on a cohort of 3,080 newly-diagnosed E-HL pts treated on 4 seminal phase 3 clinical trials (NCIC: Meyer, NEJM 2012; RAPID: Radford, NEJM 2015; EORTC-GELA H9U: Ferme, E ur J Cancer 2017; EORTC/LYSA/FIL H10: Andre, JCO 2017). External validation was done in an independent cohort of 462 E-HL pts from a large registry (Princess Margaret, Toronto). Pts with classic HL, stage I or II disease, and ages 18-65 y were included. The primary outcome was PFS. Cox models were utilized with follow-up truncated at 5 years. Baseline predictors were: sex, stage, B symptoms, histology, number & location of nodal sites, and continuous data values of age, maximum tumor diameter (MTD), white blood cell count (WBC), lymphocyte count, hemoglobin, albumin & erythrocyte sedimentation rate (ESR). Multiple imputation was used for missing data. To consider possible non-linear relationships for continuous variables we examined plots & cubic splines. We used backward elimination ( P&lt;0.1) to develop the models & internal validation with 200 bootstrap samples was conducted to estimate optimism & correct for overfitting. The final prediction equations applied optimism corrections to beta coefficients & hazard ratios (HR). Sensitivity analyses included model results stratified by PET staging (vs CT) and each HL trial. The models were evaluated in the external validation cohort & compared using c-statistics. Model scores were also grouped by tertiles for visualization in Kaplan-Meier (KM) curves. Results: Median age in the development cohort was 35y; 51% were female; 81% had nodular sclerosis & 77% stage II disease; 26% had B symptoms; 32% had mediastinal bulk (≥10cm or &gt;1/3 diameter) and the mean MTD was 6.4 cm (interquartile range (IQR), 3.9-8.2). Median follow-up was 60 months (IQR 45-75). KM estimates at 5y was 90.7% (264 events) for PFS, and 97.1% (78 events) for overall survival (OS). After backward elimination, significant variables retained were age, sex, nodal location (cervical region), MTD & albumin. Stage, ESR, or the number of nodal sites were not significant. Age was modeled with piecewise linear splines due to its non-linear relationship. See the Table for the final prediction model and optimism-corrected HRs. Optimism-corrected c-statistics in the development model was 0.63. Sensitivity analyses of stratification by type of imaging at staging or by each clinical trial demonstrated no change in model parameters or c-statistics. Most baseline characteristics and outcomes of the external validation cohort were similar besides median follow-up time (74 months, IQR 37-129). C-statistics for the E-HIPI model in external validation was 0.63. In addition, observed 5y outcomes stratified by tertile of predicted risk in the external cohort separated pts into a low & higher-risk group, which comprised 1/3 & 2/3 of pts, respectively ( Figure). Finally, model diagnostics revealed the potential for time dependency in the retained covariates. Further assessment of differential prediction between early (&lt;2y) vs. later (2-5y) PFS events is ongoing. Conclusion: Following TRIPOD methodology, we rigorously developed and externally validated the E-HIPI among &gt;3,500 E-HL pts.Discrete risk groups were delineated, and we identified several novel factors, including nodal location, sex & key continuous variables that predict 5y PFS. Additional model refinement is underway, including formal calibration & external validation via other HL registries (Stanford, Mayo/Iowa SPORE). A web-based calculator to simplify application of the E-HIPI will also be presented at ASH. Future planned modeling includes integration of differing treatments & response-adapted imaging via multistate modeling to enrich individualized survival estimates and simulation modeling to predict late consequences.

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.