Biological impact of iberdomide in patients with active systemic lupus erythematosus.

OBJECTIVES: Iberdomide is a high-affinity cereblon ligand that promotes proteasomal degradation of transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). Pharmacodynamics and pharmacokinetics of oral iberdomide were evaluated in a phase 2b study of patients with active systemic lupus erythematosus (SLE). METHODS: Adults with autoantibody-positive SLE were randomised to placebo (n=83) or once daily iberdomide 0.15 mg (n=42), 0.3 mg (n=82) or 0.45 mg (n=81). Pharmacodynamic changes in whole blood leucocytes were measured by flow cytometry, regulatory T cells (Tregs) by epigenetic assay, plasma cytokines by ultrasensitive cytokine assay and gene expression by Modular Immune Profiling. RESULTS: Iberdomide exhibited linear pharmacokinetics and dose-dependently modulated leucocytes and cytokines. Compared with placebo at week 24, iberdomide 0.45 mg significantly (p<0.001) reduced B cells, including those expressing CD268 (TNFRSF13C) (-58.3%), and plasmacytoid dendritic cells (-73.9%), and increased Tregs (+104.9%) and interleukin 2 (IL-2) (+144.1%). Clinical efficacy was previously reported in patients with high IKZF3 expression and high type I interferon (IFN) signature at baseline and confirmed here in those with an especially high IFN signature. Iberdomide decreased the type I IFN gene signature only in patients with high expression at baseline (-81.5%; p<0.001) but decreased other gene signatures in all patients. CONCLUSION: Iberdomide significantly reduced activity of type I IFN and B cell pathways, and increased IL-2 and Tregs, suggesting a selective rebalancing of immune abnormalities in SLE. Clinical efficacy corresponded to reduction of the type I IFN gene signature. TRIAL REGISTRATION NUMBER: NCT03161483.

Gene expression for biodosimetry and effect prediction purposes: promises, pitfalls and future directions - key session ConRad 2021.

PURPOSE: In a nuclear or radiological event, an early diagnostic or prognostic tool is needed to distinguish unexposed from low- and highly exposed individuals with the latter requiring early and intensive medical care. Radiation-induced gene expression (GE) changes observed within hours and days after irradiation have shown potential to serve as biomarkers for either dose reconstruction (retrospective dosimetry) or the prediction of consecutively occurring acute or chronic health effects. The advantage of GE markers lies in their capability for early (1-3 days after irradiation), high-throughput, and point-of-care (POC) diagnosis required for the prediction of the acute radiation syndrome (ARS). CONCLUSIONS: As a key session of the ConRad conference in 2021, experts from different institutions were invited to provide state-of-the-art information on a range of topics including: (1) Biodosimetry: What are the current efforts to enhance the applicability of this method to perform retrospective biodosimetry? (2) Effect prediction: Can we apply radiation-induced GE changes for prediction of acute health effects as an approach, complementary to and integrating retrospective dose estimation? (3) High-throughput and point-of-care diagnostics: What are the current developments to make the GE approach applicable as a high-throughput as well as a POC diagnostic platform? (4) Low level radiation: What is the lowest dose range where GE can be used for biodosimetry purposes? (5) Methodological considerations: Different aspects of radiation-induced GE related to more detailed analysis of exons, transcripts and next-generation sequencing (NGS) were reported.

Low-cost transcriptional diagnostic to accurately categorize lymphomas in low- and middle-income countries.

Inadequate diagnostics compromise cancer care across lower- and middle-income countries (LMICs). We hypothesized that an inexpensive gene expression assay using paraffin-embedded biopsy specimens from LMICs could distinguish lymphoma subtypes without pathologist input. We reviewed all biopsy specimens obtained at the Instituto de Cancerología y Hospital Dr. Bernardo Del Valle in Guatemala City between 2006 and 2018 for suspicion of lymphoma. Diagnoses were established based on the World Health Organization classification and then binned into 9 categories: nonmalignant, aggressive B-cell, diffuse large B-cell, follicular, Hodgkin, mantle cell, marginal zone, natural killer/T-cell, or mature T-cell lymphoma. We established a chemical ligation probe-based assay (CLPA) that quantifies expression of 37 genes by capillary electrophoresis with reagent/consumable cost of approximately $10/sample. To assign bins based on gene expression, 13 models were evaluated as candidate base learners, and class probabilities from each model were then used as predictors in an extreme gradient boosting super learner. Cases with call probabilities < 60% were classified as indeterminate. Four (2%) of 194 biopsy specimens in storage <3 years experienced assay failure. Diagnostic samples were divided into 70% (n = 397) training and 30% (n = 163) validation cohorts. Overall accuracy for the validation cohort was 86% (95% confidence interval [CI]: 80%-91%). After excluding 28 (17%) indeterminate calls, accuracy increased to 94% (95% CI: 89%-97%). Concordance was 97% for a set of high-probability calls (n = 37) assayed by CLPA in both the United States and Guatemala. Accuracy for a cohort of relapsed/refractory biopsy specimens (n = 39) was 79% and 88%, respectively, after excluding indeterminate cases. Machine-learning analysis of gene expression accurately classifies paraffin-embedded lymphoma biopsy specimens and could transform diagnosis in LMICs.

Role of a high throughput biodosimetry test in treatment prioritization after a nuclear incident.

PURPOSE: We introduce and evaluate a high throughput biodosimetry test system (REDI-Dx) suitable for testing of thousands of potential radiation victims following a mass scale nuclear event caused by detonation of a nuclear device or a nuclear accident, as part of an overall strategy for effective medical management of the crisis. MATERIALS AND METHODS: The performance of a high throughput biodosimetry test was evaluated by collecting samples of both non-irradiated presumed healthy donors as well as irradiated subjects collected as part of either cancer treatment regimens or banked from previous studies. The test measures the gene expression of a set of radiation responsive genes based on the DxDirect® genomic platform. The potential diagnostic accuracy of REDI-Dx was evaluated as a predictor of actual dose of radiation. While the REDI-Dx test has been calibrated to provide a quantitative measure of actual absorbed dose, we compared the performance of the REDI-Dx test (sensitivity and specificity) as a qualitative result at the most commonly applied thresholds 2.0 Gy and 6.0 Gy. RESULTS: The test demonstrated high specificity and lack of effect of medical conditions. Using receiver operating characteristic (ROC) curve analysis, REDI-Dx was shown to be a good predictor of actual dose for determining treatment category based on either 2.0 or 6.0 Gy, with a 98.5% sensitivity and 90% specificity for 2.0 Gy, and 92% sensitivity and 84% specificity for 6.0 Gy. Results were reproducible between clinical laboratories with an SD of 0.2 Gy for samples ≤2.0 Gy and a CV of 10.3% for samples from 2.0 to 10.0 Gy. CONCLUSIONS: Use of a biodosimetry test, like REDI-Dx test system would provide valuable information that would improve the ability to assign patients to the correct treatment category when combined with currently available biodosimetry tools, as compared to the use of existing tools alone. The REDI-Dx biodosimetry test system is for investigational use only in the U.S.A. The performance characteristics of this product have not been established.

A novel technology for multiplex gene expression analysis directly from whole blood samples stabilized at ambient temperature using an RNA-stabilizing buffer.

We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 µL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 µL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification.

A translatable predictor of human radiation exposure.

Terrorism using radiological dirty bombs or improvised nuclear devices is recognized as a major threat to both public health and national security. In the event of a radiological or nuclear disaster, rapid and accurate biodosimetry of thousands of potentially affected individuals will be essential for effective medical management to occur. Currently, health care providers lack an accurate, high-throughput biodosimetric assay which is suitable for the triage of large numbers of radiation injury victims. Here, we describe the development of a biodosimetric assay based on the analysis of irradiated mice, ex vivo-irradiated human peripheral blood (PB) and humans treated with total body irradiation (TBI). Interestingly, a gene expression profile developed via analysis of murine PB radiation response alone was inaccurate in predicting human radiation injury. In contrast, generation of a gene expression profile which incorporated data from ex vivo irradiated human PB and human TBI patients yielded an 18-gene radiation classifier which was highly accurate at predicting human radiation status and discriminating medically relevant radiation dose levels in human samples. Although the patient population was relatively small, the accuracy of this classifier in discriminating radiation dose levels in human TBI patients was not substantially confounded by gender, diagnosis or prior exposure to chemotherapy. We have further incorporated genes from this human radiation signature into a rapid and high-throughput chemical ligation-dependent probe amplification assay (CLPA) which was able to discriminate radiation dose levels in a pilot study of ex vivo irradiated human blood and samples from human TBI patients. Our results illustrate the potential for translation of a human genetic signature for the diagnosis of human radiation exposure and suggest the basis for further testing of CLPA as a candidate biodosimetric assay.

Functionalized Rhodium Intercalators for DNA Recognition.

A series of rhodium complexes containing the phenanthrenequinone diimine (phi) ligand have been prepared which bind DNA by intercalation and, upon photoactivation, promote DNA strand breaks. In this series, the ancillary, nonintercalating bipyridyl or phenanthroline ligands have been functionalized to yield complexes containing guanidinium, amido, or amino groups arranged with defined stereochemistry for site-specific interaction with the DNA bases. Lambda-1-[Rh(MGP)(2)phi](5+) (MGP = 4-(guanidylmethyl)-1,10-phenanthroline) site-specifically targets the 6-base pair sequence 5'-CATATG-3' with a binding affinity of 1 (+/-0.5) x 10(8) M(-)(1) while Delta-1-[Rh(MGP)(2)phi](5+) displays an affinity of 5 (+/-2) x 10(7) M(-)(1) for 5'-CATCTG-3'. Even though these two isomers target sites which differ by only a single base, binding is highly enantioselective. The specificity is derived chiefly from interactions of the pendant guanidinium groups with the DNA bases. For the racemates of 1-[Rh(GEB)(2)phi](5+) (GEB = (4-(2-guanidylethyl)-4'-methyl-2,2'-bipyridine) and 1-[Rh(GPB)(2)phi](5+) (GPB = (4-(2-guanidylpropyl)-4'-methyl-2,2'-bipyridine), photocleavage patterns also show the strongest site of photocleavage as 5'-CATCTG-3', the target site for Delta-1-[Rh(MGP)(2)phi](5+). Moreover, consistent with the dominance of the guanidinium groups in establishing specificity, significantly enhanced photocleavage is evident for the 1-positional isomer of these complexes, where the guanidinium moieties are directed toward the DNA (above and below the phi ligand) compared to the 2-isomer, in which the guanidinium groups are directed away from the DNA. In contrast to Lambda-1-[Rh(MGP)(2)phi](5+), Lambda-1-[Rh(GEB)(2)phi](5+) shows little cleavage at 5'-CATATG-3'; this sensitivity to linker length likely depends on the mode of recognition of 5'-CATATG-3' involving sequence-dependent unwinding of the DNA site. Analogous site-specificity or isomer-specificity is not evident with the complexes which contain pendant amido or amino functionalities. Instead these complexes appear to resemble the parent, unfunctionalized [Rh(phen)(2)phi](3+) with respect to recognition. Pendant guanidinium functionalities appear to be particularly advantageous in the construction of small molecules which bind DNA with site-specificity.