Signature Research (United States)

Despite various novel therapeutic possibilities for patients with muscle-invasive bladder cancer (MIBC), therapy response varies highly. Circulating tumor DNA (ctDNA) represents a fraction of cell-free DNA released into the bloodstream by apoptotic cell turnover or cell death in the tumor. We investigated whether ctDNA measurements before and after radical cystectomy are associated with disease recurrence. We prospectively collected plasma, tumor tissue, and germline samples before and after radical cystectomy. Seventy patients had complete data for both pre- and postsurgery analyses. Commonly mutated genes in plasma and tissue, particularly TERT and TP53, were assessed using the AVENIO ctDNA platform (research use only; Roche, Branchburg, NJ, USA) and then evaluated for a potential association with patient outcome measures, including survival and disease recurrence. In the overall study population, patients with negative ctDNA status in postsurgery plasma had significantly longer recurrence-free survival than those with positive ctDNA status (p=0.01). The ctDNA positivity fell from 46% before surgery to 23% after surgery, and a positive postoperative result predicted recurrence independently. Patients who were ctDNA positive before surgery and converted to ctDNA negative after surgery showed longer survival than those remaining ctDNA positive (median survival 36 vs 18 mo). Our results highlight that perioperative ctDNA status is associated with patient prognosis in MIBC.

Protocols for deriving midbrain dopaminergic (mDA) neurons for Parkinson’s disease (PD) modeling and therapy remain incompletely benchmarked against in vivo references. To establish transcriptomic standards, we generated an integrated human fetal whole-brain atlas and a midbrain subatlas. Whole-brain analysis revealed strong region-specific signatures, underscoring the need for global mapping before refined midbrain annotation. We implemented this two-tier strategy, BrainSTEM (Brain Single-cell Two tiEr Mapping), to systematically reassess published single-cell datasets of human midbrain culture models. BrainSTEM confirmed the presence of bona fide midbrain cell types (“on-target”), but also revealed substantial populations aligning with nonmidbrain regions (“off-target”), inflating reported mDA yields across protocols. This unbiased framework enables rigorous evaluation of differentiation outcomes, clarifies current limitations of midbrain-directed models, and provides a foundation for refining protocols toward more faithful in vitro systems for PD research and regenerative applications.

Abstract Lysosomal dysfunction and mitochondrial health are intricately connected, playing essential roles in cellular homeostasis. Lysosomes are acidic membrane-bound organelles responsible for degrading and recycling cellular waste, while mitochondria generate the energy required for cellular functions. Growing evidence implicates roles for lysosomal and mitochondrial dysfunction in neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. With novel therapeutics targeting both the lysosomal and mitochondrial functions, robust assays for compound screening are becoming critical to evaluate modulation of both organelles in disease-relevant cellular models. Here, we investigated human fibroblasts derived from healthy donors, as well as patients with Alzheimer’s and Parkinson’s disease, to assess their capacity to model key aspects of lysosomal and mitochondrial dysfunction. Lysosomal function was evaluated using various assays, including quantification of lysosomal proteins (TMEM175 and LAMP1), LysoTracker™ staining, measurement of lysosomal pH, and lysosomal enzymatic activity. Autophagic flux was assessed by measuring p62 levels as a marker of autophagy. Mitochondrial function was investigated by measuring mitochondrial calcium levels, membrane potential, oxidative stress, and mitochondrial content using MitoTracker™. To explore the potential of using human fibroblastsfor in vitrocompound screening, we validated these assays in a 384-well high-throughput format using compounds such as chloroquine and ammonium chloride. Our findings demonstrate that human fibroblasts faithfully recapitulate lysosomal and mitochondrial dysfunctions characteristic of neurodegenerative diseases. Moreover, the use of robust assays positions these cells as a valuable platform for high-throughput screening to identify novel therapeutics targeting lysosomal and mitochondrial pathways.

C. Lutterbeck and D. Kaufman equally contributing author. Introduction: Follicular lymphoma (FL) is an indolent disease with favorable outcome with antibody-based immunochemotherapy treatment. However, a subset of patients (pts) experiencing progression within 24 months have poor prognosis. Available clinicogenetic scores show suboptimal performance for predicting early progression (Jurinovic, Blood 2016). We previously showed the potential of ctDNA to identify high risk pts in a pilot from the GALLIUM trial (NCT 01332968, Bottos, ASH 2023). Here, we report an updated analysis on an expanded cohort confirming the robust prognostic value of ctDNA at screening for pts with untreated FL. Methods: We sequenced additional 200 pts to be analyzed in combination with the 216 of the pilot cohort. The new samples were selected using random stratified sampling to ensure adequate coverage of the ctDNA cohort with the GALLIUM trial population with respect to progression-free survival (PFS), FLIPI, and treatment arm. The high risk group was defined as pts with progressive diseases or death within 24 months from randomization. Targeted NGS was performed using a modified version of the AVENIO NHL analysis workflow (Roche; Research Use Only). ctDNA was quantified as mutant molecules per milliliter (MMPM) based on somatic single nucleotide variants (SNV). Results: ctDNA analysis at screening in 416 pts with FL showed a median of 102 SNVs and MMPM of 110.1. A significant correlation between MMPM and clinical prognostic markers was observed; higher MMPM was associated with larger SPD, higher FLIPI, FLIPI2, and m7-FLIPI scores (all p < 0.0001). m7-FLIPI, calculated with ctDNA derived mutations, was prognostic for pts treated with CHOP/CVP (HR [95% CI] = 1.9 [1.3,2.5]) but not with bendamustine (benda) (HR [95% CI] = 1.2 [0.81,1.6]), consistent with results from tumor sequencing (Jurinovic, ASH 2021). MMPM was a stronger marker to predict high risk (area under the receiver operating characteristic curve, AUROC = 0.67) than FLIPI (AUROC = 0.57), FLIPI2 (AUROC = 0.60) and SPD (AUROC = 0.60, Figure 1A). In a multivariate model MMPM increased the AUROC of FLIPI/2 and SPD respectively. However, MMPM as a univariate marker had the highest AUROC and in a Cox proportional hazard model pts with high MMPM experienced significantly shorter PFS (HR [95% CI] = 2.2 [1.8, 2.6], Figure 1B). Notably, the prognostic value was maintained in pts treated with CHOP/CVP (HR [95% CI] = 2.7 [2.0, 3.5]), and benda (HR [95% CI] = 1.9 [1.5, 2.5]), increasing the applicability as prognostication tool. Molecular characterization of pts with MMPM high versus low will be presented. Conclusions: In this expanded analysis, we demonstrated that ctDNA at screening is prognostic for the identification of high risk pts with untreated FL. In this large dataset, MMPM adds prognostic value beyond known factors that is maintained on CHOP/CVP and benda regimen. These results warrant additional investigation for ctDNA as a stratification/selection tool in clinical research. Research funding declaration: Genentech/Roche Keywords: genomics, epigenomics, and other -omics; liquid biopsy; diagnostic and prognostic biomarkers Potential sources of conflict of interest: C. Lutterbeck Employment or leadership position: Signature Diagnostics (Roche) D. Kaufman Employment or leadership position: Genentech (Roche) Stock ownership: Roche C. R. Bolen Employment or leadership position: Genentech (Roche) Stock ownership: Roche M. Shin Employment or leadership position: Roche A. Knapp Employment or leadership position: Roche T. Nielsen Employment or leadership position: Roche Stock ownership: Roche O. Weigert Consultant or advisory role: Roche Other remuneration: research support from InCyte A. Davies Employment or leadership position: Nil Consultant or advisory role: Roche, BMS, Kite/Gilead, Sobi, Incyte, Abbvie, Genmab Stock ownership: Nil Honoraria: AstraZeneca Educational grants: Roche Other remuneration: Research funding: AstraZeneca, MSD, Roche E. Penuel Employment or leadership position: Genentech (Roche) Stock ownership: Roche P. E. Bogard Employment or leadership position: Roche Stock ownership: Roche A. Bottos Employment or leadership position: Roche Stock ownership: Roche

7042 Background: In recent years, bispecific T cell Engagers (BsTCE) targeting CD3 and CD20 have emerged as a potent new class of therapeutics for NHL; however, a subset of patients still experience relapsed or refractory disease. Patients undergoing treatment with BsTCEs could benefit from longitudinal screening via liquid biopsy to identify baseline (primary) and treatment-induced (acquired) resistance mutations and tailor treatment accordingly. Here we present the results from a pilot study screening patients treated with Glofitamab (CD20xCD3) using a single gene Primer Extension Target Enrichment (PETE) strategy for MS4A1 (CD20 gene) in liquid biopsies. Methods: Our pilot study was conducted using libraries from the plasma of 134/155 patients with relapsed or refractory large B cell lymphoma (r/r LBCL) who underwent Glofitamab treatment at approved dose in the phase I/II trial NP30179 ( NCT03075696 ) (Dickinson, et al. N Engl J Med 2022), and paired PBMC/PDB available in 91 cases. Primers were designed against the coding regions of the MS4A1 gene. Enrichment was performed using a workflow optimized for the detection of somatic variants in cell-free DNA isolated from plasma. Results were analyzed using a modified AVENIO circulating tumor (ct) DNA (Roche; For Research Use Only) analysis workflow. Variants detected by the analysis pipeline were further filtered based on inclusion criteria: allele fraction &gt; 0.1%, rarity in the cohort, impact on protein function in silico , and if sample was available, absence in germline. Tumor burden as assessed by ctDNA was obtained from retrospective sequencing data. Response to treatment was assessed by PET/CT using the Lugano Criteria. Results: Using inclusion criteria a total of 11/134 (8.2%) patients were identified with a total of 16 unique MS4A1 candidate mutations at baseline or during treatment. The Best Overall Investigator Response (BOR) was progressive disease (PD) for 7 patients and partial metabolic response (PR) for 4 patients. All patients with BOR PR experienced disease progression before treatment completion. Sufficient samples were available to demonstrate expansion of the candidate mutation by the end of treatment (EOT) timepoint for 5 patients. Four of the identified mutations were previously reported in the literature, the remaining mutations were novel, and in vitro characterization demonstrated their functional impact. Conclusions: This work identifies known and novel mutations in CD20 in plasma samples as a potential contributing mechanism to relapsed or refractory cases of NHL. Although the prevalence appears to be low, this is consistent with previous reports and supports investigation of the clinical utility, including utility as a potential predictive biomarker, of sequencing this gene during treatment with CD20xCD3 BsTCE. Future and ongoing work will screen additional cohorts and therapy combinations.

Chronic kidney disease (CKD) represents a global health issue with a high socioeconomic impact. Beyond a progressive decline of kidney function, patients with CKD are at increased risk of cardiovascular diseases, including heart failure (HF) and sudden cardiac death. HF in CKD can manifest both as HF with reduced ejection fraction and HF with preserved ejection fraction, with the latter further increasing in relative importance in the more advanced stages of CKD. Typical cardiac remodeling characteristics in uremic cardiomyopathy include left ventricular hypertrophy, myocardial fibrosis, cardiac electrical dysregulation, capillary rarefaction, and microvascular dysfunction, which are triggered by increased cardiac preload, cardiac afterload, and preload and afterload-independent factors. The pathophysiological mechanisms underlying cardiac remodeling in CKD are multifactorial and include neurohormonal activation (with increased activation of the renin-angiotensin-aldosterone system, the sympathetic nervous system, and mineralocorticoid receptor signaling), cardiac steroid activation, mitochondrial dysfunction, inflammation, innate immune activation, and oxidative stress. Furthermore, disturbances in cardiac metabolism and calcium homeostasis, macrovascular and microvascular dysfunction, increased cellular profibrotic responses, the accumulation of uremic retention solutes, and mineral and bone disorders also contribute to cardiovascular disease and HF in CKD. Here, we review the current knowledge of HF in CKD, including the clinical characteristics and pathophysiological mechanisms revealed in animal studies. We also elaborate on the detrimental impact of comorbidities of CKD on HF using hypertension as an example and discuss the clinical characteristics of hypertensive heart disease and the genetic predisposition. Overall, this review aims to increase the understanding of HF in CKD to support future research and clinical translational approaches for improved diagnosis and therapy of this vulnerable patient population.

The generation of forensic DNA profiles consisting of single nucleotide polymorphisms (SNPs) is now being facilitated by wider adoption of next-generation sequencing (NGS) methods in casework laboratories. At the same time, and in part because of this advance, there is an intense focus on the generation of SNP profiles from evidentiary specimens for so-called forensic or investigative genetic genealogy (FGG or IGG) applications. However, FGG methods are constrained by the algorithms for genealogical database searches, which were designed for use with single-source profiles, and the fact that many forensic samples are mixtures. To enable the use of two-person mixtures for FGG, we developed a workflow, MixDeR, for the deconvolution of mixed SNP profiles. MixDeR, a flexible and easy to use R package and Shiny app, processes ForenSeq Kintelligence® (QIAGEN, Inc.) SNP genotyping results and directs deconvolution of the profiles in EuroForMix (EFM). MixDeR then filters the EFM outputs to produce inferred single-source genotypes in reports formatted for use with GEDmatch® PRO. An optional MixDeR output includes metrics that assist with testing and validation of the workflow. As the Shiny app provides a graphical user interface and the software is designed to be run offline, MixDeR should be suitable for use by any laboratory developing FGG capabilities, no matter their bioinformatic resources or expertise.

Malgré une coopération initiale, le projet de parc éolien offshore en baie de Saint-Brieuc a suscité une forte opposition de la part des pêcheurs. Restituant 20 ans d’itinéraire concertatif et conflictuel, nous analysons ce glissement vers le conflit tout en mettant en lumière des points de bascule et le rôle des proximités. Ce qui est en jeu, c’est la territorialisation d’une technologie innovante et de l’intérêt général. Cette étude nous invite à discuter la notion d’acceptation sociale, en prenant en compte le potentiel effet boomerang de l’appropriation, pourtant souvent présentée comme le niveau le plus élevé de l’acceptation. Sans cesse mise à l’épreuve, l’acceptation est notamment déterminée par les dynamiques des proximités et de la confiance.