Glioblastoma (GBM)'s median overall survival is almost 21 months. Six phase 3 immunotherapy clinical trials have recently been published, yet 5/6 did not meet approval by regulatory bodies. For the sixth, approval is uncertain. Trial failures result from multiple factors, ranging from intrinsic tumor biology to clinical trial design. Understanding the clinical and basic science of these 6 trials is compelled by other immunotherapies reaching the point of advanced phase 3 clinical trial testing. We need to understand more of the science in human GBMs in early trials: the "window of opportunity" design may not be best to understand complex changes brought about by immunotherapeutic perturbations of the GBM microenvironment. The convergence of increased safety of image-guided biopsies with "multi-omics" of small cell numbers now permits longitudinal sampling of tumor and biofluids to dissect the complex temporal changes in the GBM microenvironment as a function of the immunotherapy.

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers with limited treatment options. Nearly all PDAC harbors a KRAS mutation, and oncogenic KRAS signaling is a key driver. Localization of the KRAS protein to the plasma membrane is absolutely required for downstream tumorigenic activities, making trafficking of KRAS to the plasma membrane a compelling target for treatment. In a familial pancreatic cancer syndrome with a striking five cases of PDAC, we recently discovered a rare, inherited gain-of-function mutation in the gene RABL3 that impacts the kinetics of KRAS intracellular trafficking. RABL3 was found to indirectly interact with KRAS, and the familial RABL3 mutation accelerates trafficking of KRAS to the plasma membrane. Here, we present pre-clinical experiments evaluating the therapeutic potential of targeting RABL3 to disrupt KRAS trafficking and activity. To achieve inducible knockdown of RABL3, we have engineered KPCY cells (tumor cells derived from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre; Rosa26YFP/YFP mice) to express a doxycycline-inducible shRNA targeting RABL3. In vitro, RABL3 knockdown results in robust suppression of KPCY cell proliferation. Moreover, the knockdown of RABL3 abrogated tumor growth of KPCY cells in immune-competent subcutaneous and orthotopic pancreatic cancer mouse models. Single-cell transcriptome profiling of tumors revealed immune infiltration of T cells, B cells, macrophages, and dendritic cells following RABL3 knockdown, suggesting that RABL3 knockdown creates vulnerabilities seen with suppression of KRAS activity. Validating these findings in a human context, we find a significantly elevated level of RABL3 expression across multiple human PDAC cell lines, consistent with a driver role in PDAC and a critical role in regulating KRAS trafficking. Similarly, analysis of human datasets revealed elevated RABL3 transcript levels in PDAC TCGA specimens compared to matched normal tissue as well as in PDAC patient-derived organoids compared to the GTEx cohort of normal pancreas tissues. Furthermore, there is a trend for worse patient survival with higher RABL3 expression levels. Therefore, to evaluate the potential of RABL3 knockdown to improve the potency, efficacy, and toxicity when combined with other PDAC therapeutic targets, we are currently testing the impacts of RABL3 knockdown on the therapeutic profile of newly available small-molecular KRAS inhibitors. Our findings provide pre-clinical support for targeting RABL3 to disrupt KRAS intracellular trafficking and inhibit KRAS activity for the treatment of PDAC. Citation Format: Woo-Jeong Jeong, Katherine J. Aney, Ethan Chen, Annan Yang, Giselle A. Uribe, Andrew Aguirre, Sahar Nissim. Targeting RABL3 to disrupt KRAS trafficking and activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A104.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA- sequencing approach in which tissue is reversibly fi xed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate fi ndings using GeoMx whole transcriptome atlas profiling, fi ling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition fi nition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies fi es changes in all major cell types during injury and recovery. We define fi ne the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, fi ltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. fi ltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

Background Xalnesiran, a small interfering RNA molecule that targets a conserved region of the hepatitis B virus (HBV) genome and silences multiple HBV transcripts, may have efficacy, with or without an immunomodulator, in patients with chronic HBV infection. Methods We conducted a phase 2, multicenter, randomized, controlled, adaptive, open-label platform trial that included the evaluation of 48 weeks of treatment with xalnesiran at a dose of 100 mg (group 1), xalnesiran at a dose of 200 mg (group 2), xalnesiran at a dose of 200 mg plus 150 mg of ruzotolimod (group 3), xalnesiran at a dose of 200 mg plus 180 mu g of pegylated interferon alfa-2a (group 4), or a nucleoside or nucleotide analogue (NA) alone (group 5) in participants with chronic HBV infection who had virologic suppression with NA therapy. The primary efficacy end point was hepatitis B surface antigen (HBsAg) loss (HBsAg level, <0.05 IU per milliliter) at 24 weeks after the end of treatment. Safety was also assessed. Download a PDF of the Plain Language Summary. Results Among 159 participants (30, 30, 34, 30, and 35 in groups 1 through 5, respectively), the primary end-point event occurred in 7% (95% confidence interval [CI], 1 to 22) of those in group 1, in 3% (95% CI, 0 to 17) of those in group 2, in 12% (95% CI, 3 to 28) of those in group 3, in 23% (95% CI, 10 to 42) of those in group 4, and in none (95% CI, 0 to 10) of those in group 5. In groups 1 through 5, respectively, HBsAg seroconversion occurred in 3%, none, 3%, 20%, and none of the participants at 24 weeks after the end of treatment. HBsAg loss with or without seroconversion occurred only in participants with a screening HBsAg level below 1000 IU per milliliter. In groups 1 through 5, respectively, grade 3 or 4 adverse events occurred in 17%, 10%, 18%, 50%, and 6% of the participants, with the most frequent event being an elevated alanine aminotransferase level. Conclusions Among participants with chronic HBV infection who had virologic suppression with NA therapy, treatment with xalnesiran plus an immunomodulator resulted in HBsAg loss at 24 weeks after the end of treatment in a substantial percentage of participants. Grade 3 or 4 adverse events were not uncommon.

Abstract Pancreatic acinar cells are responsible for producing large amounts of digestive enzymes, which are essential for nutrient breakdown but make it difficult to capture the transcriptome of individual cells. To address this challenge, we use a reversible fixative that is compatible with 10X Genomics, thereby preserving the acinar transcriptome and capturing cell composition more faithful to histology. Using this method, we transcriptionally characterize all cell types in the healthy mouse pancreas, and track how these populations change during acute pancreatitis and subsequent recovery. We identify immune cells in the healthy and inflamed pancreas at a level of detail previously unappreciated, characterizing T cell subsets including Th2, γδT, and regulatory T cells. Pancreatitis elicits an influx of neutrophils along with activation and proliferation of macrophages and dendritic cells. Observing significant transcriptional changes in acinar cells after pancreatitis, we define an Acinar to Ductal Metaplasia Index (ADMI), a gene signature that can be used to measure deviation from normal acinar identity in bulk or single cell RNA datasets. We corroborate ADMI using orthogonal GeoMx data, and validate its utility in independent published datasets. We are now expanding our study to perform transcriptomics using our fixation method on pancreatic cancer precursor lesions. Together, we aim to characterize cell types in the mouse pancreas under healthy, inflamed, and precursor conditions to establish a reference atlas for future research on the exocrine pancreas in healthy and diseased states. Citation Format: Katherine J. Aney, Woo-Jeong Jeong, Andres F. Vallejo, Ethan Chen, Austin Wang, Stephanie K. Dougan, Kellie Wise, Kirk Jensen, Luciano Martelotto, Sahar Nissim. A novel approach for exocrine pancreas transcriptomics reveals the cellular landscape of the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A066.

Background/Aims Four-week treatment of linvencorvir (RO7049389) was generally safe and well tolerated, and showed anti-viral activity in chronic hepatitis B (CHB) patients. This study evaluated the efficacy, safety, and pharmacokinetics of 48-week treatment with linvencorvir plus standard of care (SoC) in CHB patients. Methods This was a multicentre, non-randomized, non-controlled, open-label phase 2 study enrolling three cohorts: nucleos(t)ide analogue (NUC)-suppressed patients received linvencorvir plus NUC (Cohort A, n=32); treatment-naïve patients received linvencorvir plus NUC without (Cohort B, n=10) or with (Cohort C, n=30) pegylated interferon-α (Peg-IFN-α). Treatment duration was 48 weeks, followed by NUC alone for 24 weeks. Results 68 patients completed the study. No patient achieved functional cure (sustained HBsAg loss and unquantifiable HBV DNA). By Week 48, 89% of treatment-naïve patients (10/10 Cohort B; 24/28 Cohort C) reached unquantifiable HBV DNA. Unquantifiable HBV RNA was achieved in 92% of patients with quantifiable baseline HBV RNA (14/15 Cohort A, 8/8 Cohort B, 22/25 Cohort C) at Week 48 along with partially sustained HBV RNA responses in treatment-naïve patients during follow-up period. Pronounced reductions in HBeAg and HBcrAg were observed in treatment-naïve patients, while HBsAg decline was only observed in Cohort C. Most adverse events were grade 1–2, and no linvencorvir-related serious adverse events were reported. Conclusions 48-week linvencorvir plus SoC was generally safe and well tolerated, and resulted in potent HBV DNA and RNA suppression. However, 48-week linvencorvir plus NUC with or without Peg-IFN did not result in the achievement of functional cure in any patient.

AimsRO7049389 (linvencorvir) is a developmental oral treatment for chronic hepatitis B virus infection. The aim of this work was to conduct mass balance (MB) and absolute bioavailability (BA) analyses in healthy volunteers, alongside in vitro evaluations of the metabolism of RO7049389 and a major circulating active metabolite M5 in human hepatocytes, and physiologically based pharmacokinetic (PBPK) modelling to refine the underlying drug disposition paradigm.MethodsParticipants in the clinical study (MB: Caucasian, male, n = 6; BA: Caucasian and Asian, male and female, n = 16, 8 in each ethnic groups) received oral [14C] or unlabelled RO7049389 (600/1000 mg) followed by 100 μg intravenous [13C]RO7049389. Metabolic pathways with fractions metabolized—obtained from the in vitro incubation results of 10 μM [14C]RO7049389 and 1 μM M5 with (long‐term cocultured) human hepatocytes in the absence and presence of the cytochrome P450 3A4 (CYP3A4) inhibitor itraconazole—were used to complement the PBPK models, alongside the clinical MB and BA data.ResultsThe model performance in predicting the pharmacokinetic profiles of RO7049389 and M5 aligned with clinical observations in Caucasians and was also successfully applied to Asians. Accordingly, the drug disposition pathways for RO7049389 were postulated with newly characterized estimates of the fractions: biliary excretion by P‐glycoprotein (~41%), direct glucuronidation via uridine 5′‐diphosphoglucuronosyltransferase 1A3 (~11%), hexose conjugation (~6%), oxidation by CYP3A4 (~28%) and other oxidation reactions (~9%).ConclusionThese results support the ongoing clinical development program for RO7049389 and highlight the broader value of PBPK and MB analyses in drug development.

Introduction: In the Phase 3 IMbrave150 trial (NCT03434379), atezolizumab + bevacizumab demonstrated a clinically meaningful survival benefit over sorafenib in patients with unresectable hepatocellular carcinoma (HCC), including those with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. We used IMbrave150 data to investigate the safety and risk of viral reactivation or flare in infected patients treated with atezolizumab + bevacizumab or sorafenib. Methods: Patients with unresectable HCC not previously treated with systemic therapy were randomized 2:1 to atezolizumab + bevacizumab or sorafenib. In this exploratory analysis, safety was continually evaluated, including for hepatic adverse events. Patients were monitored for HBV and HCV reactivation and flare at screening, the beginning of Cycles 5 and 9, and at treatment discontinuation. Results: Of 501 enrolled patients, 485 were included in the safety population; 329 (68%) received atezolizumab + bevacizumab, and 156 (32%) received sorafenib. Overall, 150 (31%) and 58 (12%) patients had HBV and HCV infections, respectively. The safety profiles of atezolizumab + bevacizumab and sorafenib were consistent across patients, regardless of viral infection. Overall, hepatic serious adverse events occurred in 11% of patients receiving atezolizumab + bevacizumab and 8% receiving sorafenib. HBV or HCV reactivation occurred in 2% or 16% of atezolizumab + bevacizumab-treated patients, respectively, versus 7% or 14% with sorafenib. There were no instances of hepatitis flare with atezolizumab + bevacizumab. Conclusions: Atezolizumab + bevacizumab had a similar hepatic safety profile in patients with and without HBV or HCV infection. Viral reactivation rates were similar between arms. Overall, these data support the use of atezolizumab + bevacizumab in patients with HCC and HBV or HCV infection without any special precaution.

Background Toll-like receptor 7 (TLR7) agonists augment immune activity and have potential for the treatment of chronic hepatitis B virus (HBV) infection. We aimed to assess the safety and tolerability of RO7020531 (also called RG7854), a prodrug of the TLR7 agonist RO7011785, in healthy volunteers and patients with chronic HBV infection.Methods This randomised, observer-blind, placebo-controlled, phase 1 study was done in two parts. Part 1 was done at one site in New Zealand and part 2 was done at 12 sites in Bulgaria, Hong Kong, Italy, New Zealand, the Netherlands, Taiwan, Thailand, and the UK. In part 1, healthy volunteers were randomly assigned (4:1) within one of eight dose cohorts (3 mg, 10 mg, 20 mg, 40 mg, 60 mg, 100 mg, 140 mg, or 170 mg) to receive a single RO7020531 dose or placebo or randomly assigned (4:1) within one of three dose cohorts (100 mg, 140 mg, or 170 mg) to receive either RO7020531 or placebo every other day for 13 days. In part 2, nucleoside or nucleotide analogue-suppressed patients with chronic HBV infection were randomly assigned (4:1) within cohorts 1-3 (150 mg, 150 mg, or 170 mg) to receive either RO7020531 or placebo and treatment-naive patients with chronic HBV infection were randomly assigned (3:1) in cohort 4 to receive either 150 mg of RO7020531 or placebo. Patients were treated every other day for 6 weeks. Study medication was administered orally to participants after they had fasted. Study participants and investigational staff were masked to treatment allocation. The primary outcome was the safety and tolerability of RO7020531, as measured by the incidence and severity of adverse events and the incidence of laboratory, vital sign, and electrocardiogram abnormalities, and was analysed in all participants who received at least one dose of the study medication. This trial is registered with ClinicalTrials.gov, NCT02956850, and the study is complete.Findings Between Dec 12, 2016, and March 21, 2021, 340 healthy volunteers were screened in part 1, of whom 80 were randomly assigned in the single ascending dose study (eight assigned RO7020531 in each cohort and 16 assigned placebo) and 30 were randomly assigned in the multiple ascending dose study (eight assigned RO7020531 in each cohort and six assigned placebo), and 110 patients were screened in part 2, of whom 30 were randomly assigned in cohorts 1-3 (16 assigned RO7020531 150 mg, eight assigned RO7020531 170 mg, and six assigned placebo) and 20 were randomly assigned in cohort 4 (15 assigned RO7020531 and five assigned placebo). All randomly assigned participants received at least one dose of a study drug and were included in the safety analysis. All tested doses of RO7020531 were safe and had acceptable tolerability in healthy volunteers and patients. The most frequent treatment-related adverse events among the total study population were headache (15 [9%] of 160 participants), influenza-like illness (seven [4%] of 160 participants), and pyrexia (ten [6%] of 160 participants). Most adverse events were mild and transient. There were no severe or serious adverse events in healthy volunteers. In the patient cohorts, there was one severe adverse event (influenza-like illness with 170 mg of RO7020531) and one serious adverse event (moderate influenza-like illness with a 3-day hospitalisation in a treatment-naive patient receiving RO7020531). There were no treatment-related deaths.Interpretation Due to acceptable safety and tolerability, RO7020531 should continue to be developed for the treatment of patients with chronic HBV infection. Funding F Hoffmann-La Roche.Copyright (c) 2022 Published by Elsevier Ltd. All rights reserved.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM) 1 , 2 . Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110—an oncolytic herpes virus (oHSV) 3 . In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue 4 . These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi -omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi -omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.

favorable preclinical profile for clinical advancement.

Germline genetic variants implicated in increasing lifetime risk of pancreatic cancer (PDAC) have been identified in ∼4% to 10% of cases. Clinical features such as family history have poor sensitivity in identifying carriers of these risk variants. Genetic testing for these germline variants has potential to guide risk assessment and surveillance recommendations in high-risk individuals to promote prevention and early detection measures. Furthermore, identification of novel germline variants can offer important insights into pathogenesis that may inform precision medicine approaches. This article reviews current understanding of germline mutations associated with PDAC risk and implications of genetic testing.

spontaneously; of the remaining 91 patients, only five were on treatment. Conclusion:Our preliminary results are highly promising.Once refined and validated, the algorithm will allow us to identify patients in need of HBV outreach and care leading to evaluation for hepatitis D as well.It will also help to highlight clinical settings with suboptimal HBV care in our healthcare system.Applicable to other health systems utilizing EPIC EMR, our algorithm will be vital in streamlining case finding, freeing up resources for patient engagement and care, and advancing viral hepatitis elimination.