High CD34-positive cell dose in matched unrelated donor allogeneic hematopoietic stem cell transplant is not associated with graft-versus-host disease or mortality.

BACKGROUND: CD34+ stem cells serve as the primary graft source for allogeneic transplants, with a minimum of 2-4 × 106 cells/kg needed for engraftment. There are conflicting data on outcomes at high stem cell doses, with studies limited by few patients receiving doses far above the minimum target. STUDY DESIGN AND METHODS: In this retrospective, single-center study of patients with hematologic malignancies who underwent matched unrelated donor transplants, we assessed outcomes for engraftment, survival, relapse, and graft-versus-host disease (GVHD) for the highest CD34+ dose quintile (>13 × 106 cells/kg, n = 36) compared to the remaining patients (n = 139). Similar analysis was performed correlating T cell dose and outcomes. RESULTS: There was no difference between the groups in neutrophil engraftment, with a trend toward faster platelet engraftment. There was no significant difference in mortality (adjusted risk ratio [aRR] = 1.02, 95% confidence interval [CI] = 0.85-1.22), relapse (aRR = 1.10, 95% CI = 0.85-1.42), or overall survival by Kaplan-Meier analysis (p = .44). High CD34+ dose was not associated with higher incidence of acute GVHD (aRR = 0.99 grades II-IV, aRR = 1.18 grades III-IV) or chronic GVHD (aRR = 0.87 overall, RR = 1.21 severe). There was limited correlation between CD34+ and T cell dose (R2 = .073), and there was no significant difference in survival, relapse, or GVHD in the highest T cell dose quintile (n = 33) compared to the remaining quintiles (n = 132). DISCUSSION: We found no difference in survival, relapse, or GVHD incidence or severity in patients receiving CD34+ doses above prior cutoffs reported in the literature. These data do not support the routine use of graft CD34+ dose reduction.

Updated COVID-19 clearance time among patients with cancer in the Delta and Omicron waves.

BACKGROUND: COVID-19 infection delays therapy and in-person evaluation for oncology patients, but clinic clearance criteria are not clearly defined. METHODS: We conducted a retrospective review of oncology patients with COVID-19 at a tertiary care center during the Delta and Omicron waves and compared clearance strategies. RESULTS: Median clearance by two consecutive negative tests was 32.0 days (Interquartile Range [IQR] 22.0-42.5, n = 153) and was prolonged in hematologic malignancy versus solid tumors (35.0 days for hematologic malignancy, 27.5 days for solid tumors, p = 0.01) and in patients receiving B-cell depletion versus other therapies. Median clearance by single negative test was reduced to 23.0 days (IQR 16.0-33.0), with recurrent positive rate 25.4% in hematologic malignancy versus 10.6% in solid tumors (p = 0.02). Clearance by a predefined waiting period required 41 days until an 80% negative rate. CONCLUSIONS: COVID-19 clearance remains prolonged in oncology patients. Single-negative test clearance can balance delays in care with risk of infection in patients with solid tumors.

t(4;12)(q12;p13) ETV6-rearranged AML without eosinophilia does not involve PDGFRA: relevance for imatinib insensitivity.

Acute myeloid leukemia (AML) with t(4;12)(q12;p13) translocation is rare and often associated with an aggressive clinical course and poor prognosis. Previous reports based on fluorescence in situ hybridization (FISH) analysis have suggested that ETV6::PDGFRA fusions are present in these patients, despite the absence of eosinophilia, which is typically found in other hematopoietic malignancies with PDGFRA-containing fusions. We first detected an ETV6-SCFD2 fusion by targeted RNA sequencing in a patient with t(4;12)(q12;p13) who had been diagnosed with an ETV6-PDGFRA fusion by FISH analysis but failed to respond to imatinib. We then retrospectively identified 4 additional patients with AML and t(4;12)(q12;p13) with apparent ETV6-PDGFRA fusions using chromosome and FISH analysis and applied targeted RNA sequencing to archival material. We again detected rearrangements between ETV6 and non-PDGFRA 4q12 genes, including SCFD2, CHIC2, and GSX2. None of the 3 patients who received imatinib based on the incorrect assumption of an ETV6-PDGFRA fusion responded. Our findings highlight the importance of using a sequencing-based assay to confirm the presence of targetable gene fusions, particularly in genomic regions, such as 4q12, with many clinically relevant genes that are too close to resolve by chromosome or FISH analysis. Finally, combining our data and review of the literature, we show that sequence-confirmed ETV6-PDGFRA fusions are typically found in eosinophilic disorders (3/3 cases), and patients with t(4;12)(q12;p13) without eosinophilia are found to have other 4q12 partners on sequencing (17/17 cases).

Acalabrutinib, venetoclax, and obinutuzumab as frontline treatment for chronic lymphocytic leukaemia: a single-arm, open-label, phase 2 study.

BACKGROUND: Both continuous therapy with acalabrutinib and fixed-duration therapy with venetoclax-obinutuzumab are effective for previously untreated chronic lymphocytic leukaemia. We hypothesised that frontline time-limited, minimal residual disease (MRD)-guided triplet therapy with acalabrutinib, venetoclax, and obinutuzumab would induce deep (ie, more patients with undetectable MRD) and durable remissions. METHODS: In this open-label, single-arm, investigator-sponsored, phase 2 study, patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma were recruited from two academic hospitals in Boston, MA, USA. Eligible patients were aged 18 years or older, with an Eastern Cooperative Oncology Group performance status of 0-2, and were treatment naive. Patients were treated in 28 day cycles. Acalabrutinib monotherapy was given orally at 100 mg twice daily for cycle 1, then combined for six cycles with intravenous obinutuzumab (100 mg on cycle 2 day 1, 900 mg on day 2, 1000 mg on day 8, and 1000 mg on day 15 and on day 1 of cycles 3-7); and from the beginning of cycle 4, oral venetoclax was dosed daily using an accelerated ramp-up from 20 mg on day 1 to 400 mg by day 22 and continued at this dose thereafter. Patients continued on acalabrutinib 100 mg twice daily and venetoclax 400 mg once daily until day 1 of cycle 16 or day 1 of cycle 25. If the patient had undetectable MRD in the bone marrow they were given the option to discontinue therapy at the start of cycle 16 (if also in complete remission) or at the start of cycle 25 (if at least in partial remission). The primary endpoint was complete remission with undetectable MRD in the bone marrow (defined as <1 chronic lymphocytic leukaemia cell per 10 000 leucocytes as measured by four-colour flow cytometry), at cycle 16 day 1. Safety and activity endpoints were assessed in all patients who received at least one dose of any study drug. This study is registered with ClinicalTrials.gov, NCT03580928, and is ongoing. FINDINGS: Between Aug 2, 2018, and May 23, 2019, 37 patients with chronic lymphocytic leukaemia were enrolled and all received at least one dose of any study drug. The median age of patients was 63 years (IQR 57-70), and ten (27%) were female and 27 (73%) were male. Median follow-up was 27·6 months (IQR 25·1-28·2). At cycle 16 day 1, 14 (38% [95% CI 22-55]) of 37 participants had a complete remission with undetectable MRD in the bone marrow. The most common grade 3 or 4 haematological adverse event was neutropenia (16 [43%] of 37 patients). The most common grade 3-4 non-haematological adverse events were hyperglycaemia (three [8%]) and hypophosphataemia (three [8%]). Serious adverse events occurred in nine (24%) patients; the most common was neutropenia in three (8%) patients. There have been no deaths on study. INTERPRETATION: Acalabrutinib, venetoclax, and obinutuzumab is a highly active and well tolerated frontline therapy for chronic lymphocytic leukaemia. Although the primary endpoint of this study was not met, the high proportion of patients who had undetectable MRD in the bone marrow supports further investigation of this regimen, which is being tested against acalabrutinib-venetoclax and chemoimmunotherapy in an ongoing phase 3 study (NCT03836261). FUNDING: AstraZeneca and a Dana-Farber Cancer Institute Collaborative Award.

Leukemia vaccine overcomes limitations of checkpoint blockade by evoking clonal T cell responses in a murine acute myeloid leukemia model.

We have developed a personalized vaccine whereby patient derived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T cell response against shared and neo-antigens. We postulated that the dendritic cell (DC)/AML fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen specific lymphocytes that would provide a critical substrate for checkpoint blockade mediated activation. Using an immunocompetent murine leukemia model, we examined the immunologic response and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor specific immunity. Mice treated with checkpoint blockade alone had rapid leukemia progression and demonstrated only a modest extension of survival. Vaccination with DC/AML fusions resulted in the expansion of tumor specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long term survivors exhibited increased T cell clonal diversity and were resistant to subsequent tumor challenge. The combined DC/AML fusion vaccine and checkpoint blockade treatment offers unique synergy inducing the durable activation of leukemia specific immunity, protection from lethal tumor challenge and the selective expansion of tumor reactive clones.

A Hybrid Extracellular Electron Transfer Pathway Enhances the Survival of Vibrio natriegens.

Vibrio natriegens is the fastest-growing microorganism discovered to date, making it a useful model for biotechnology and basic research. While it is recognized for its rapid aerobic metabolism, less is known about anaerobic adaptations in V. natriegens or how the organism survives when oxygen is limited. Here, we describe and characterize extracellular electron transfer (EET) in V. natriegens, a metabolism that requires movement of electrons across protective cellular barriers to reach the extracellular space. V. natriegens performs extracellular electron transfer under fermentative conditions with gluconate, glucosamine, and pyruvate. We characterized a pathway in V. natriegens that requires CymA, PdsA, and MtrCAB for Fe(III) citrate and Fe(III) oxide reduction, which represents a hybrid of strategies previously discovered in Shewanella and Aeromonas Expression of these V. natriegens genes functionally complemented Shewanella oneidensis mutants. Phylogenetic analysis of the inner membrane quinol dehydrogenases CymA and NapC in gammaproteobacteria suggests that CymA from Shewanella diverged from Vibrionaceae CymA and NapC. Analysis of sequenced Vibrionaceae revealed that the genetic potential to perform EET is conserved in some members of the Harveyi and Vulnificus clades but is more variable in other clades. We provide evidence that EET enhances anaerobic survival of V. natriegens, which may be the primary physiological function for EET in VibrionaceaeIMPORTANCE Bacteria from the genus Vibrio occupy a variety of marine and brackish niches with fluctuating nutrient and energy sources. When oxygen is limited, fermentation or alternative respiration pathways must be used to conserve energy. In sedimentary environments, insoluble oxide minerals (primarily iron and manganese) are able to serve as electron acceptors for anaerobic respiration by microorganisms capable of extracellular electron transfer, a metabolism that enables the use of these insoluble substrates. Here, we identify the mechanism for extracellular electron transfer in Vibrio natriegens, which uses a combination of strategies previously identified in Shewanella and Aeromonas We show that extracellular electron transfer enhanced survival of V. natriegens under fermentative conditions, which may be a generalized strategy among Vibrio spp. predicted to have this metabolism.

Double-Hit and Triple-Hit Follicular Lymphoma.

OBJECTIVES: To better characterize the clinicopathologic presentation and outcomes of follicular lymphoma with MYC and BCL2 and/or BCL6 rearrangements (double-hit and triple-hit follicular lymphoma), we present three cases from our institution and perform a literature review of 37 published cases. METHODS: Cases were identified using institutional SoftPath software and the MEDLINE database via the PubMed search engine. Clinical and pathologic data were collected with subsequent stratification by histologic grade and treatment for comparison. RESULTS: Similar to classic follicular lymphoma, patients presented most often with low-grade (1-2) but high-stage (III-IV) disease with absence of B symptoms; however, overall survival was worse than that of traditional follicular lymphoma. In a small sample size, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R) achieved better outcomes than a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Specific pathologic features that might prompt testing for MYC rearrangement include elevated proliferation index out of proportion to cytology and aggressive features such as angioinvasion. CONCLUSIONS: Double-hit and triple-hit follicular lymphoma may be better classified as a distinct entity from classical follicular lymphoma with a worse prognosis. Aggressive therapy with a treatment regimen used for high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements might be beneficial, but more evidence is needed to justify aggressive treatment as standard of care.

Characterization of resistance to a potent D-peptide HIV entry inhibitor.

BACKGROUND: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket. RESULTS: Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data. CONCLUSIONS: These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

Hypomethylating agent alters the immune microenvironment in acute myeloid leukaemia (AML) and enhances the immunogenicity of a dendritic cell/AML vaccine.

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.

Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens.

A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries ( e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 ± 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E. coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V. natriegens-derived CFPS platform is versatile and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.

Dendritic Cell Therapies for Hematologic Malignancies.

Dendritic cells (DCs) are potent antigen-presenting cells that constitute a major component of the immune system's role in the recognition, elimination, and tolerance of cancer. The unique immunologic capabilities of DCs have recently been harnessed for therapeutic use with the creation of DC-based anti-tumor vaccines, several of which have moved into testing in clinical trials for hematologic malignancies. This review summarizes how treatment strategies using DC-based anti-tumor vaccines are advancing immunotherapeutic options for these diseases.

Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.

The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.

'We need to have as many Americans covered as possible'.

Unable to influence debate in the House of Representatives on the American Health Care Act, lobbyists and trade group executives are hoping for a more receptive ear in the Senate. "It really is a new day," said Chip Kahn, CEO of the Federation of American Hospitals, alluding to the fact that key Senate leaders have said they are writing their own bill. Among the federation's top priorities, Kahn said, is addressing Medicare cuts enacted in the Affordable Care Act, especially if a new law results in dramatic increases in the uninsured. Kahn recently spoke with Modern Healthcare Managing Editor Matthew Weinstock about the health reform debate, as well as Kahn's call to streamline quality measures. The following is an edited transcript.

Providers' AHCA worries rise after latest CBO analysis.

The Congressional Budget Office estimated that 23 million people would lose health coverage by 2026 under the American Health Care Act.

Vibrio natriegens as a fast-growing host for molecular biology.

A rapidly growing bacterial host would be desirable for a range of routine applications in molecular biology and biotechnology. The bacterium Vibrio natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min. We report the development of genetic tools and methods to engineer V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes.

Intracranial hemorrhage in cancer patients treated with anticoagulation.

Both venous thromboembolism and intracranial metastases are common complications in the setting of primary brain tumors and metastatic malignancies. Anticoagulation is indicated in the presence of cancer-associated thrombosis in order to limit the risk of pulmonary embolism; however, there is reluctance to initiate anticoagulation in the setting of intracranial metastatic disease due to potential for intracranial hemorrhage. Recent evidence suggests that therapeutic anticoagulation can be safely administered in the setting of metastatic brain tumors. This review examines the current understanding of the pathophysiology of intracranial hemorrhage in malignancy, describes the incidence of intracranial hemorrhage in the setting of brain tumors with therapeutic anticoagulation, and outlines management strategies relevant to the treatment of intracranial hemorrhage in the setting of anticoagulation.