Integrated genetic, epigenetic, and immune landscape of TP53 mutant AML and higher risk MDS treated with azacitidine.

Background: TP53 mutations are associated with an adverse prognosis in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (HR-MDS). However, the integrated genetic, epigenetic, and immunologic landscape of TP53-mutated AML/HR-MDS is not well defined. Objectives: To define the genetic, epigenetic, and immunologic landscape of TP53-mutant and TP53 wild-type AML and HR-MDS patients. Design: Post hoc analysis of TP53-mutant and TP53 wild-type patients treated on the randomized FUSION trial with azacitidine ± the anti-PD-L1 antibody durvalumab. Methods: We performed extensive molecular, epigenetic, and immunologic assays on a well-annotated clinical trial dataset of 61 patients with TP53-mutated disease (37 AML, 24 MDS) and 144 TP53 wild-type (89 AML, 55 MDS) patients, all of whom received azacitidine-based therapy. A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed. DNA methylation arrays, immunophenotyping and immune checkpoint expression by flow cytometry, and gene expression profiles by bulk RNA sequencing were assessed at baseline and serially during the trial. Results: Global DNA methylation from peripheral blood was independent of TP53 mutation and allelic status. AZA therapy led to a statistically significant decrease in global DNA methylation scores independent of TP53 mutation status. In BM from TP53-mutant patients, we found both a higher T-cell population and upregulation of inhibitory immune checkpoint proteins such as PD-L1 compared to TP53 wild-type. RNA sequencing analyses revealed higher expression of the myeloid immune checkpoint gene LILRB3 in TP53-mutant samples suggesting a novel therapeutic target. Conclusion: This integrated analysis of the genetic, epigenetic, and immunophenotypic landscape of TP53 mutant AML/HR-MDS suggests that differences in the immune landscape resulting in an immunosuppressive microenvironment rather than epigenetic differences contribute to the poor prognosis of TP53-mutant AML/HR-MDS with mono- or multihit TP53 mutation status. Trial registration: FUSION trial (NCT02775903).

Discovery and Preclinical Activity of BMS-986351, an Antibody to SIRPα That Enhances Macrophage-mediated Tumor Phagocytosis When Combined with Opsonizing Antibodies.

In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). SIGNIFICANCE: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.

Exploration of Tumor Biopsy Gene Signatures to Understand the Role of the Tumor Microenvironment in Outcomes to Lisocabtagene Maraleucel.

In the TRANSCEND NHL 001 study, 53% of patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with lisocabtagene maraleucel (liso-cel) achieved a complete response (CR). To determine characteristics of patients who did and did not achieve a CR, we examined the tumor biology and microenvironment from lymph node tumor biopsies. LBCL biopsies from liso-cel-treated patients were taken pretreatment and ∼11 days posttreatment for RNA sequencing (RNA-seq) and multiplex immunofluorescence (mIF). We analyzed gene expression data from pretreatment biopsies (N = 78) to identify gene sets enriched in patients who achieved a CR to those with progressive disease. Pretreatment biopsies from month-3 CR patients displayed higher expression levels of T-cell and stroma-associated genes, and lower expression of cell-cycle genes. To interpret whether LBCL samples were "follicular lymphoma (FL)-like," we constructed an independent gene expression signature and found that patients with a higher "FL-like" gene expression score had longer progression-free survival (PFS). Cell of origin was not associated with response or PFS, but double-hit gene expression was associated with shorter PFS. The day 11 posttreatment samples (RNA-seq, N = 73; mIF, N = 53) had higher levels of chimeric antigen receptor (CAR) T-cell densities and CAR gene expression, general immune infiltration, and immune activation in patients with CR. Further, the majority of T cells in the day 11 samples were endogenous. Gene expression signatures in liso-cel-treated patients with LBCL can inform the development of combination therapies and next-generation CAR T-cell therapies.

A randomized phase 2 trial of azacitidine with or without durvalumab as first-line therapy for older patients with AML.

Evidence suggests that combining immunotherapy with hypomethylating agents may enhance antitumor activity. This phase 2 study investigated the activity and safety of durvalumab, a programmed death-ligand 1 (PD-L1) inhibitor, combined with azacitidine for patients aged ≥65 years with acute myeloid leukemia (AML), including analyses to identify biomarkers of treatment response. Patients were randomized to first-line therapy with azacitidine 75 mg/m2 on days 1 through 7 with (Arm A, n = 64) or without (Arm B, n = 65) durvalumab 1500 mg on day 1 every 4 weeks. Overall response rate (complete response [CR] + CR with incomplete blood recovery) was similar in both arms (Arm A, 31.3%; Arm B, 35.4%), as were overall survival (Arm A, 13.0 months; Arm B, 14.4 months) and duration of response (Arm A, 24.6 weeks; Arm B, 51.7 weeks; P = .0765). No new safety signals emerged with combination treatment. The most frequently reported treatment-emergent adverse events were constipation (Arm A, 57.8%; Arm B, 53.2%) and thrombocytopenia (Arm A, 42.2%; Arm B, 45.2%). DNA methylation, mutational status, and PD-L1 expression were not associated with response to treatment. In this study, first-line combination therapy with durvalumab and azacitidine in older patients with AML was feasible but did not improve clinical efficacy compared with azacitidine alone. ClinicalTrials.gov: NCT02775903.

A randomized phase 2 trial of azacitidine with or without durvalumab as first-line therapy for higher-risk myelodysplastic syndromes.

Azacitidine-mediated hypomethylation promotes tumor cell immune recognition but may increase the expression of inhibitory immune checkpoint molecules. We conducted the first randomized phase 2 study of azacitidine plus the immune checkpoint inhibitor durvalumab vs azacitidine monotherapy as first-line treatment for higher-risk myelodysplastic syndromes (HR-MDS). In all, 84 patients received 75 mg/m2 subcutaneous azacitidine (days 1-7 every 4 weeks) combined with 1500 mg intravenous durvalumab on day 1 every 4 weeks (Arm A) for at least 6 cycles or 75 mg/m² subcutaneous azacitidine alone (days 1-7 every 4 weeks) for at least 6 cycles (Arm B). After a median follow-up of 15.25 months, 8 patients in Arm A and 6 in Arm B remained on treatment. Patients in Arm A received a median of 7.9 treatment cycles and those in Arm B received a median of 7.0 treatment cycles with 73.7% and 65.9%, respectively, completing ≥4 cycles. The overall response rate (primary end point) was 61.9% in Arm A (26 of 42) and 47.6% in Arm B (20 of 42; P = .18), and median overall survival was 11.6 months (95% confidence interval, 9.5 months to not evaluable) vs 16.7 months (95% confidence interval, 9.8-23.5 months; P = .74). Durvalumab-related adverse events (AEs) were reported by 71.1% of patients; azacitidine-related AEs were reported by 82% (Arm A) and 81% (Arm B). Grade 3 or 4 hematologic AEs were reported in 89.5% (Arm A) vs 68.3% (Arm B) of patients. Patients with TP53 mutations tended to have a worse response than patients without these mutations. Azacitidine increased programmed cell death ligand 1 (PD-L1 [CD274]) surface expression on bone marrow granulocytes and monocytes, but not blasts, in both arms. In summary, combining azacitidine with durvalumab in patients with HR-MDS was feasible but with more toxicities and without significant improvement in clinical outcomes over azacitidine alone. This trial was registered at www.clinicaltrials.gov as #NCT02775903.

Immunomodulation by durvalumab and pomalidomide in patients with relapsed/refractory multiple myeloma.

This study sought to understand how the programmed death ligand 1 (PD-L1) inhibitor durvalumab and the immunomodulatory agent pomalidomide regulate immune cell activation and function in patients with relapsed/refractory (RR) multiple myeloma (MM). Immunologic changes in peripheral blood and bone marrow of patients treated with durvalumab as monotherapy or in combination with pomalidomide with/without dexamethasone were characterized by assessing subsets of immune cells and gene signatures to understand the immunomodulatory effect of the treatment. Soluble PD-L1 levels were elevated at screening in patients with RRMM but did not correlate with response to durvalumab combination therapy. Immune cell subsets were increased in peripheral blood during treatment with durvalumab and pomalidomide, and combination therapy induced significant gene expression changes in the MM tumor microenvironment versus durvalumab alone. Estimation of cell populations based on RNA sequencing data revealed increased monocytes, neutrophils, and natural killer cells with the combination therapy, but not with durvalumab alone. Additionally, multiplex immunofluorescence of bone marrow demonstrated that immune populations were different in responders versus nonresponders to durvalumab plus pomalidomide with dexamethasone therapy. Overall, durvalumab effectively blocked soluble PD-L1; however, durvalumab monotherapy was not associated with immunologic changes, which were observed with combination therapy.

Validation of a Host Response Assay, SeptiCyte LAB, for Discriminating Sepsis from Systemic Inflammatory Response Syndrome in the ICU.

RATIONALE: A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility. OBJECTIVES: This study evaluated the diagnostic performance of a molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults. METHODS: The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts. MEASUREMENTS AND MAIN RESULTS: In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity. CONCLUSIONS: SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).

Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster.

The sand rat Psammomys obesus is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene Pdx1. The sequence of sand rat Pdx1 has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.

Detection of gene expression signatures related to underlying disease and treatment in rheumatoid arthritis patients.

OBJECTIVES: Gene expression signatures can provide an unbiased view into the molecular changes underlying biologically and medically interesting phenotypes. We therefore initiated this study to identify signatures that would be of utility in studying rheumatoid arthritis (RA). METHODS: We used microarray profiling of peripheral blood mononuclear cells (PBMCs) in 30 RA patients to assess the effect of different biologic agent (biologics) treatments and to quantify the degree of a type-I interferon (IFN) signature in these patients. A numeric score was derived for the quantification step and applied to patients with RA. To further characterize the IFN response in our cohort, we employed type-I IFN treatment of PBMCs in vitro and in reporter assays. RESULTS: Profiling identified a subset of RA patients with upregulation of type-I IFN-regulated transcripts, thereby corroborating previous reports showing RA to be heterogeneous for an IFN component. A comparison of individuals currently untreated with a biologic with those treated with infliximab, tocilizumab, or abatacept suggested that each biologic induces a specific gene signature in PBMCs. CONCLUSIONS: It is possible to observe signs of type-I IFN pathway activation in a subset of clinically active RA patients without C-reactive protein elevation. Furthermore, biologics-specific gene signatures in patients with RA indicate that looking for a biologic-specific response pattern may be a potential future tool for predicting individual patient response.

The role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family.

BACKGROUND: Type-I interferons, type-II interferons, and the IL-10 family are helical cytokines with similar three-dimensional folds. However, their homologous relationship is difficult to detect on the basis of sequence alone. We have previously described the discovery of the human type-III interferons (IFN lambda-1, -2, -3 or IL-29, IL-28A, IL-28B), which required a combination of manual and computational techniques applied to predicted protein sequences. PRINCIPAL FINDINGS: Here we describe how the use of gene structure analysis and comparative genomics enabled a more extensive understanding of these genes early in the discovery process. More recently, additional mammalian genome sequences have shown that there are between one and potentially nine copies of interferon lambda genes in each genome, and that several species have single exon versions of the interferon lambda gene. SIGNIFICANCE: The variable number of single exon type-I interferons in mammals, along with recently identified genes in zebrafish homologous to interferons allows a story of interferon evolution to be proposed. This model suggests that the gene duplications and single exon retrotransposons of mammalian type-III interferons are positively selected for within a genome. These characteristics are also shared with the fish interferons and could be responsible for the generation of the IL10 family and also the single exon type-I interferons.