Next-generation sequencing for measurable residual disease detection in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a blood cancer characterized by acquired genetic mutations. There is great interest in accurately establishing measurable residual disease (MRD) burden in AML patients in remission after treatment but at risk of relapse. However, inter- and intrapatient genetic diversity means that, unlike in the chronic myeloid and acute promyelocytic leukaemias, no single genetic abnormality is pathognomonic for all cases of AML MRD. Next-generation sequencing offers the opportunity to test broadly and deeply for potential genetic evidence of residual AML, and while not currently accepted for such use clinically, is likely to be increasingly used for AML MRD testing in the future.

Intramolecular C-H Functionalization Followed by a [2σ + 2π] Addition via an Intermediate Nickel-Nitridyl Complex.

Irradiation of a disphenoidal Ni(II) azido complex, [Cz tBu(Pyr iPr)2NiN3] (1), revealed an unprecedented nickel complex, [Cz tBu(Pyr iPr)(NH2-Pyr iPr)] (2), in >90% isolated yield. As evidenced by single-crystal X-ray diffraction, 2 is produced by double intramolecular C-H activation of a putative nickel-nitridyl intermediate, [Cz tBu(Pyr iPr)2Ni-⃛N•]. Calculations support the generation of an intermediate with significant nitridyl radical character after the loss of N2, which, in turn, undergoes tandem C-H activations, leading to functionalized intermediates and products. This is an unprecedented example of transient Ni-⃛N•-promoted intramolecular C-H functionalization, followed by a [2σ + 2π] addition, yielding bis-metallacyclic product 2. Complex 2 is also observed from the reaction of Ni(I) precursor Cz tBu(Pyr iPr)2Ni (3) and Me3SiN3, suggesting a unique thermal route toward a masked nickel-nitridyl intermediate.

A Series of 4- and 5-Coordinate Ni(II) Complexes: Synthesis, Characterization, Spectroscopic, and DFT Studies.

A series of four- and five-coordinate Ni(II) complexes Cz tBu(Pyr iPr)2NiX (1-3 and 1·THF-3·THF), where X = Cl, Br, and I, were synthesized and fully characterized by NMR and UV-vis spectroscopy, X-ray crystallography, cyclic voltammetry, and density functional theory calculations. The solid-state structures of 1-3 reveal rare examples of seesaw Ni(II) complexes. In solution, 1-3 bind reversibly to a THF molecule to form five-coordinate adducts. The electronic transitions in the visible region (630-680 nm), attributed to LMCT bands, for 1 → 3 exhibit a bathochromic shift. The thermochromic tendency of the five-coordinate complexes implies the loss of THF coordination at elevated temperatures. Finally, the electronic properties of all Ni(II) complexes were studied by time-dependent density functional theory calculations to characterize the nature of the excited states.

Benign Natural Progression of Small Cavernous Carotid Aneurysms Suggests Limited Clinical Utility of Serial Longitudinal Follow-up.

BACKGROUND AND OBJECTIVES: Aneurysms in the cavernous segment of the internal carotid artery (ICA) often present in an indolent fashion with limited morbidity. However, their growth progression and possible rupture over time remains poorly defined, thereby limiting optimization of serial follow-up. Thus, we aim to describe the progression of cavernous ICA aneurysms over time, as well as the patient and aneurysm characteristics associated with possible growth and rupture status. METHODS: We identified a consecutive cohort of 157 patients from 2007 to 2021 with cavernous ICA aneurysms. Patient demographic data, possible risk factors, presenting symptoms, radiographic features of aneurysms, size progression, rupture status, and concomitant noncavernous aneurysm rupture data were manually extracted. RESULTS: One hundred and fifty-seven patients (mean age at diagnosis 57.2 ± 15.6 years; 85.4% females) with 174 cavernous carotid aneurysms (CCAs) were followed for an average of 7.1 ± 4.8 years. 76.4% of aneurysms were identified incidentally, with predominantly ocular palsies as the presenting symptoms in remaining primary cases. Most aneurysms were small, and of the 168 aneurysms that were followed, 98.2% did not demonstrate appreciable growth. Of the aneurysms that grew, it took an average of 6.0 years to grow 1.6 ± 0.2 mm. Demographic data, hypertension, and smoking status were not associated with aneurysm growth. Most radiographic features also were not associated with growth, except long-axis diameter, which had an odds ratio of 1.4 (CI: 1.2, 1.8) on multivariable analysis. Presenting clinical symptoms were not associated with growth. No CCAs ruptured during follow-up. CONCLUSION: Cavernous ICA aneurysms in our series demonstrate no rupture and limited growth over years of clinical follow-up. No radiographic or patient risk factors were associated with growth except initial aneurysm size. Hence, small CCAs may not require close follow-up over time.

Labeling T Cells to Track Immune Response to Immunotherapy in Glioblastoma.

While the advent of immunotherapy has revolutionized cancer treatment, its use in the treatment of glioblastoma (GBM) has been less successful. Most studies using immunotherapy in GBM have been negative and the reasons for this are still being studied. In clinical practice, interpreting response to immunotherapy has been challenging, particularly when trying to differentiate between treatment-related changes (i.e., pseudoprogression) or true tumor progression. T cell tagging is one promising technique to noninvasively monitor treatment efficacy by assessing the migration, expansion, and engagement of T cells and their ability to target tumor cells at the tumor site.

Recurrent germline variant in ATM associated with familial myeloproliferative neoplasms.

Genetic predisposition (familial risk) in the myeloproliferative neoplasms (MPNs) is more common than the risk observed in most other cancers, including breast, prostate, and colon. Up to 10% of MPNs are considered to be familial. Recent genome-wide association studies have identified genomic loci associated with an MPN diagnosis. However, the identification of variants with functional contributions to the development of MPN remains limited. In this study, we have included 630 MPN patients and whole genome sequencing was performed in 64 individuals with familial MPN to uncover recurrent germline predisposition variants. Both targeted and unbiased filtering of single nucleotide variants (SNVs) was performed, with a comparison to 218 individuals with MPN unselected for familial status. This approach identified an ATM L2307F SNV occurring in nearly 8% of individuals with familial MPN. Structural protein modeling of this variant suggested stabilization of inactive ATM dimer, and alteration of the endogenous ATM locus in a human myeloid cell line resulted in decreased phosphorylation of the downstream tumor suppressor CHEK2. These results implicate ATM, and the DNA-damage response pathway, in predisposition to MPN.

Tandem chimeric antigen receptor (CAR) T cells targeting EGFRvIII and IL-13Rα2 are effective against heterogeneous glioblastoma.

Background: Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. Methods: Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors. Results: Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05). Conclusions: We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.

Pembrolizumab and decitabine for refractory or relapsed acute myeloid leukemia.

BACKGROUND: The powerful 'graft versus leukemia' effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic cell transplantation in acute myeloid leukemia (AML) provides rationale for investigation of immune-based therapies in this high-risk blood cancer. There is considerable preclinical evidence for potential synergy between PD-1 immune checkpoint blockade and the hypomethylating agents already commonly used for this disease. METHODS: We report here the results of 17 H-0026 (PD-AML, NCT02996474), an investigator sponsored, single-institution, single-arm open-label 10-subject pilot study to test the feasibility of the first-in-human combination of pembrolizumab and decitabine in adult patients with refractory or relapsed AML (R-AML). RESULTS: In this cohort of previously treated patients, this novel combination of anti-PD-1 and hypomethylating therapy was feasible and associated with a best response of stable disease or better in 6 of 10 patients. Considerable immunological changes were identified using T cell receptor ß sequencing as well as single-cell immunophenotypic and RNA expression analyses on sorted CD3+ T cells in patients who developed immune-related adverse events (irAEs) during treatment. Clonal T cell expansions occurred at irAE onset; single-cell sequencing demonstrated that these expanded clones were predominately CD8+ effector memory T cells with high cell surface PD-1 expression and transcriptional profiles indicative of activation and cytotoxicity. In contrast, no such distinctive immune changes were detectable in those experiencing a measurable antileukemic response during treatment. CONCLUSION: Addition of pembrolizumab to 10-day decitabine therapy was clinically feasible in patients with R-AML, with immunological changes from PD-1 blockade observed in patients experiencing irAEs.

Impact of Conditioning Intensity and Genomics on Relapse After Allogeneic Transplantation for Patients With Myelodysplastic Syndrome.

PURPOSE: Patients with myelodysplastic syndrome (MDS) are at risk of relapse after allogeneic hematopoietic cell transplantation. The utility of ultra-deep genomic testing to predict and the impact of conditioning intensity to prevent MDS relapse are unknown. METHODS: Targeted error-corrected DNA sequencing was performed on preconditioning blood samples from patients with MDS (n = 48) from the Blood and Marrow Transplant Clinical Trials Network 0901 phase III randomized clinical trial, which compared outcomes by allogeneic hematopoietic cell transplantation conditioning intensity in adult patients with < 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment. Clinical end points (53-month median follow-up) included transplant-related mortality (TRM), relapse, relapse-free survival (RFS), and overall survival (OS). Of the 48 patients examined, 14 experienced TRM, 23 are relapse-free, and 11 relapsed, of which 7 died. RESULTS: Using a previously described set of 10 gene regions, 42% of patients (n = 20) had mutations detectable before random assignment to reduced intensity conditioning (RIC) or myeloablative conditioning (MAC). Testing positive was associated with increased rates of relapse (3-year relapse, 40% v 11%; P = .022) and decreased OS (3-year OS, 55% v 79%, P = .045). In those testing positive, relapse rates were higher (3-year relapse, 75% v 17%; P = .003) and RFS was lower (3-year RFS, 13% v 49%; P = .003) in RIC versus MAC arms. Testing additional genes, including those associated with MDS, did not improve prognostication. CONCLUSION: This study provides evidence that targeted DNA sequencing in patients with MDS before transplant can identify those with highest post-transplant relapse rates. In those testing positive, random assignment to MAC lowered but did not eliminate relapse risk.

Personalized Single-Cell Proteogenomics to Distinguish Acute Myeloid Leukemia from Non-Malignant Clonal Hematopoiesis.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.

Synthesis, characterization, DFT calculations, and reactivity study of a nitrido-bridged dimeric vanadium(iv) complex.

Two vanadium(iii) complexes, CztBu(PyriPr)2VCl2 (1) and CztBu(PyriPr)2V(N3)2 (2), were synthesized and characterized. Chemical reduction of both 1 and 2 gives the thermally stable nitrido-bridged vanadium(iv) dimer complex, [{CztBu(PyriPr)2}V]2(µ-N)2 (3), which is a rare example of a dimeric vanadium(iv) complex bridged by two nitrido ligands. The nitride ligands of 3 are unreactive due to the well-protected environment provided by the pincer ligand and its substituents, as is supported by its X-ray crystal structure and further described by DFT calculations.

Impact of Conditioning Intensity of Allogeneic Transplantation for Acute Myeloid Leukemia With Genomic Evidence of Residual Disease.

PURPOSE: Patients with acute myeloid leukemia (AML) in remission remain at risk for relapse even after allogeneic hematopoietic cell transplantation (alloHCT). AML measurable residual disease (MRD) status before alloHCT has been shown to be prognostic. Whether modulation of the intensity of the alloHCT conditioning regimen in patients with AML who test positive for MRD can prevent relapse and improve survival is unknown. METHODS: Ultra-deep, error-corrected sequencing for 13 commonly mutated genes in AML was performed on preconditioning blood from patients treated in a phase III clinical trial that randomly assigned adult patients with myeloid malignancy in morphologic complete remission to myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC). RESULTS: No mutations were detected in 32% of MAC and 37% of RIC recipients; these groups had similar survival (3-year overall survival [OS], 56% v 63%; P = .96). In patients with a detectable mutation (next-generation sequencing [NGS] positive), relapse (3-year cumulative incidence, 19% v 67%; P < .001) and survival (3-year OS, 61% v 43%; P = .02) was significantly different between the MAC and RIC arms, respectively. In multivariable analysis for NGS-positive patients, adjusting for disease risk and donor group, RIC was significantly associated with increased relapse (hazard ratio [HR], 6.38; 95% CI, 3.37 to 12.10; P < .001), decreased relapse-free survival (HR, 2.94; 95% CI, 1.84 to 4.69; P < .001), and decreased OS (HR, 1.97; 95% CI, 1.17 to 3.30; P = .01) compared with MAC. Models of AML MRD also showed benefit for MAC over RIC for those who tested positive. CONCLUSION: This study provides evidence that MAC rather than RIC in patients with AML with genomic evidence of MRD before alloHCT can result in improved survival.

Highly multiplexed proteomic assessment of human bone marrow in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by abnormal clonal proliferation of myeloid progenitor cells found predominantly within the bone marrow (BM) and blood. Recent studies suggest that genetic and phenotypic alterations in the BM microenvironment support leukemogenesis and allow leukemic cells to survive and evade chemotherapy-induced death. However, despite substantial evidence indicating the role of tumor-host interactions in AML pathogenesis, little is known about the complex microenvironment of the BM. To address this, we performed novel proteomic profiling of the noncellular compartment of the BM microenvironment in patients with AML (n = 10) and age- and sex-matched healthy control subjects (n = 10) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). We show that proteomic assessment of blood or RNA-sequencing of BM are suboptimal alternate screening strategies to determine the true proteomic composition of the extracellular soluble compartment of AML patient BM. Proteomic analysis revealed that 168 proteins significantly differed in abundance, with 91 upregulated and 77 downregulated in leukemic BM. A highly connected signaling network of cytokines and chemokines, including IL-8, was found to be the most prominent proteomic signature associated with AML in the BM microenvironment. We report the first description of significantly elevated levels of the myelosuppressive chemokine CCL23 (myeloid progenitor inhibitory factor-1) in both AML and myelodysplastic syndrome patients and perform functional experiments supportive of a role in the suppression of normal hematopoiesis. This unique paired RNA-sequencing and proteomics data set provides innovative mechanistic insights into AML and healthy aging and should serve as a useful public resource.