Survival outcomes of patients newly diagnosed with diffuse large B-cell lymphoma: real-world evidence from a German claims database.

PURPOSE: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma with increasing incidence. Although the burden of disease is high, only limited current real-world data on survival analysis, especially survival time, of German patients with DLBCL are available. This retrospective claims-based analysis was conducted to describe real-world survival evidence and treatment patterns of patients with DLBCL in Germany. METHODS: Using a large claims database of the German statutory health insurance with 6.7 million enrollees, we identified patients between 2010 and 2019 who were newly diagnosed with DLBCL (index date) and had no other cancer co-morbidity. Overall survival (OS) from index date and from the end of each treatment line was plotted by means of the Kaplan-Meier estimator, both for the overall cohort and stratified by treatment regimen. Treatment lines were identified based on a predefined set of medications categorized by established DLBCL treatment recommendations. RESULTS: 2495 incident DLBCL patients were eligible for the study. After index date, 1991 patients started a first-line, 868 a second-line, and 354 a third-line therapy. In first line, 79.5% of patients received a Rituximab-based therapy. 5.0% of the of the 2495 patients received a stem cell transplantation. Overall, median OS after index was 96.0 months. CONCLUSION: DLBCL-associated mortality is still high, especially in relapsed patients and in the elderly. Therefore, there is a high medical need for new effective treatments that can improve survival outcomes in DLBCL patients.

Healthcare Resource Utilization and Associated Costs of German Patients with Diffuse Large B-Cell Lymphoma: A Retrospective Health Claims Data Analysis.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma with increasing prevalence. Although the disease burden associated with DLBCL is high, only limited data on healthcare resource utilization (HCRU) and associated costs of German patients with DLBCL is available. METHODS: Using a large claims database of the German statutory health insurance with 6.7 million enrollees, we identified patients who were newly diagnosed with DLBCL between 2011 and 2018 (index date). Treatment lines were identified based on a predefined set of medication. HCRU and related costs were collected for the entire post index period and per treatment line. RESULTS: A total of 2495 incident DLBCL patients were eligible for the analysis. The average follow-up time after index was 41.7 months. During follow-up, 1991 patients started a first-line treatment, 868 a second-line treatment, and 354 a third-line treatment. Overall, patients spent on average (SD) 5.24 (6.17) days per month in hospital after index. While on anti-cancer treatment, this number increased to nine (10.9) in first-line, 8.7 (13.7) in second-line, and 9.4 (15.8) in third-line treatments. Overall costs per patient per month (PPPM) increased from €421 (875.70) before to €3695 (4652) after index. While on a treatment line, PPPM costs were €17,170 (10,246) in first-line, €13,362 (12,685) in second-line, and €12,112 (16,173) in third-line treatments. Time-unadjusted absolute costs sum up to €59,868 (43,331), €35,870 (37,387), and €28,832 (40,540) during first-line, second-line, and third-line treatments, respectively. The main cost drivers were hospitalizations (71% of total costs) and drug acquisition costs (18% of total costs). CONCLUSIONS: The financial burden of DLBCL in Germany is high, mainly due to hospitalization and drug costs. Therefore, there is a high medical need for new cost-effective therapeutic options that can lower the disease burden and remain financially viable to support the growing number of patients with this aggressive disease.

Central amygdala circuit dynamics underlying the benzodiazepine anxiolytic effect.

Benzodiazepines (BZDs) have been a standard treatment for anxiety disorders for decades, but the neuronal circuit interactions mediating their anxiolytic effect remain largely unknown. Here, we find that systemic BZDs modulate central amygdala (CEA) microcircuit activity to gate amygdala output. Combining connectome data with immediate early gene (IEG) activation maps, we identified the CEA as a primary site for diazepam (DZP) anxiolytic action. Deep brain calcium imaging revealed that brain-wide DZP interactions shifted neuronal activity in CEA microcircuits. Chemogenetic silencing showed that PKCδ+/SST- neurons in the lateral CEA (CEAl) are necessary and sufficient to induce the DZP anxiolytic effect. We propose that BZDs block the relay of aversive signals through the CEA, in part by local binding to CEAl SST+/PKCδ- neurons and reshaping intra-CEA circuit dynamics. This work delineates a strategy to identify biomedically relevant circuit interactions of clinical drugs and highlights the critical role for CEA circuitry in the pathophysiology of anxiety.

Spectral recording of gene expression history by fluorescent timer protein.

Monitoring spatio-temporal patterns of gene expression by fluorescent proteins requires longitudinal observation, which is often difficult to implement. Here, we fuse a fluorescent timer (FT) protein with an immediate early gene (IEG) promoter to track live gene expression in single cells. This results in a stimulus- and time-dependent spectral shift from blue to red for subsequent monitoring with fluorescence activated cell sorting (FACS) and live cell imaging. This spectral shift enables imputing the time point of activity post-hoc to dissociate early and late responders from a single snapshot in time. Thus, we provide a tool for tracking stimulus-driven IEG expression and demonstrate proof of concept exploiting promoter::FT fusions, adding new dimensions to experiments that require reconstructing spatio-temporal patterns of gene expression in cells, tissues or living organisms.

Correlation of Cytomegalovirus (CMV) Disease Severity and Mortality With CMV Viral Burden in CMV-Seropositive Donor and CMV-Seronegative Solid Organ Transplant Recipients.

BACKGROUND: The rate of cytomegalovirus (CMV) viral load increase and peak viral loads are associated with CMV disease in kidney and liver transplant recipients, but relationships to disease severity or mortality have not been shown. METHODS: Using stored serial serum specimens from renal (n = 59) and liver (n = 35) transplant recipients (D+R-; CMV-seropositive donors, CMV-seronegative recipients) from 2 prospective, randomized, controlled, interventional prophylaxis trials of CMV immune globulin (CMVIG), CMV viral load was measured using the COBAS quantitative polymerase chain reaction assay and the World Health Organization CMV standard. Patients with severe CMV-associated disease were classified according to trial definitions. Pairwise comparisons of mean viral load among deceased, surviving diseased, and nondiseased patients were analyzed by 2-way analysis of variance. To determine if viral load could predict mortality, receiver operating characteristic (ROC) curves were constructed using area under the curve (AUC) of the viral load and peak viral concentration (Vmax). RESULTS: Viral load (mean log10 [AUC], peak viral load [Vmax]) for patients with severe CMV disease was significantly higher compared with nondiseased patients (P < .001). Similarly, higher viral burden was significantly associated with mortality (P < .001). Viral load AUC and Vmax AUROCs for predicting mortality were 0.796 and 0.824, respectively, for renal patients, and 0.769 and 0.807, respectively, for liver patients. CONCLUSIONS: Using specimens from studies preceding the antiviral prophylaxis era, CMV viral load was associated with severe CMV disease and death, supporting CMV viral load quantification as a proxy for CMV disease severity and disease-associated mortality end points in solid organ transplantation.

Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia.

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.

Acute infections, cost and time to reporting of HIV test results in three U.S. State Public Health Laboratories.

OBJECTIVE: In three U.S. State Public Health Laboratories (PHLs) using a fourth-generation immunoassay (IA), an HIV-1/HIV-2 differentiation antibody IA and a nucleic acid test (NAT), we characterized the yield and time to reporting of acute infections, and cost per positive specimen. METHODS: Routine HIV testing data were collected from July 1, 2012-June 30, 2013 for Massachusetts and Maryland PHLs, and from November 27, 2012-June 30, 2013 for Michigan PHL. Massachusetts and Michigan used fourth-generation and differentiation IAs with NAT conducted by a referral laboratory. In Maryland, fourth-generation IA repeatedly reactive specimens were followed by a Western blot (WB), and those with negative or indeterminate results were tested with a differentiation IA and HIV-1 NAT, and if positive by NAT, confirmed by a different HIV-1 NAT. Specimens from WB-positive persons at risk for HIV-2 were tested with a differentiation IA and, if positive, with an HIV-2 WB and/or differential HIV-1/HIV-2 proviral DNA polymerase chain reaction. RESULTS: Among 7914 specimens from Massachusetts PHL, 6069 from Michigan PHL, and 36,266 from Maryland PHL, 0.10%, 0.02% and 0.05% acute infections were identified, respectively. Massachusetts and Maryland PHLs each had 1 HIV-2 positive specimen. The median time from specimen receipt to laboratory reporting of results for acute infections at Massachusetts, Michigan and Maryland PHLs was 8, 11, and 7 days respectively. The laboratory cost per HIV positive specimen was $336 (Massachusetts), $263 (Michigan) and $210 (Maryland). CONCLUSIONS: Acute and established infections were found by PHLs using fourth-generation IA in conjunction with antibody tests and NAT. Time to reporting of acute HIV test results to clients was suboptimal, and needs to be streamlined to expedite treatment and interrupt transmission.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death.

Acute Infections, Cost per Infection and Turnaround Time in Three United States Hospital Laboratories Using Fourth-Generation Antigen-Antibody Human Immunodeficiency Virus Immunoassays.

Background. To improve clinical and public health outcomes through early human immunodeficiency virus (HIV) detection, fourth-generation antigen/antibody immunoassay (4IA) and supplemental testing results must be returned rapidly. Methods. We examined HIV testing data at Harborview Medical Center (HMC), Massachusetts General Hospital (MGH), and the Medical University of South Carolina (MUSC), which used 4IA and supplemental antibody and nucleic acid tests (NATs). At MGH and MUSC, HIV-1 Western blot (WB) and HIV-2 testing were conducted at a reference laboratory. We compared time from specimen collection to laboratory result for established (positive WB) and acute infections (reactive 4IA, negative/indeterminate WB, detectable NAT), and we calculated testing cost per positive-test result. Results. From 3731 (MUSC) to 19 774 (MGH) tests were conducted; 0.01% (MGH) to 0.05% (HMC) were acute infections. Each laboratory had reactive 4IA, WB-negative, or indeterminate specimens without NAT (ie, potential acute infections). Time to result was 1.5 (HMC) to 5.2 days (MGH) for acute and 1.0 (HMC) to 5.2 days (MGH) for established infections. Costs were $1054 (MGH) to $1521 (MUSC). Conclusions. Conducting supplemental testing in-house lowered turnaround times, which may be further reduced with rapid HIV-1/HIV-2 differentiation tests. Hospitals may benefit from quantitative NATs not requiring physician orders, so all potential acute infections receive NAT.

Nucleic acid testing by public health referral laboratories for public health laboratories using the U.S. HIV diagnostic testing algorithm.

BACKGROUND: Many public health laboratories adopting the U.S. HIV laboratory testing algorithm do not have a nucleic acid test (NAT), which is needed when the third- or fourth-generation HIV screening immunoassay is reactive and the antibody-based supplemental test is non-reactive or indeterminate. OBJECTIVES: Among public health laboratories utilizing public health referral laboratories for NAT conducted as part of the algorithm, we evaluated the percentage of screening immunoassays needing NAT, the number of specimens not meeting APTIMA (NAT) specifications, time to APTIMA result, the proportion of acute infections (i.e., reactive APTIMA) among total infections, and screening immunoassay specificity. STUDY DESIGN: From August 2012 to April 2013, 22 laboratories enrolled to receive free APTIMA (NAT) at New York or Florida public health referral laboratories. Data were analyzed for testing conducted until June 2013. RESULTS: Submitting laboratories conducted a median of 4778 screening immunoassays; 0-1.3% (median 0.2%) needed NAT. Of 140 specimens received, 9 (6.4%) did not meet NAT specifications. The median time from specimen collection to reporting the 11 reactive NAT results was ten days, including six days from receipt in the submitting laboratory to shipment to the referral laboratory. Acute infections ranged from 0 to 12.5% (median 0%) of total infections. Third- and fourth-generation immunoassays met package insert specificity values. CONCLUSIONS: Public health referral laboratories provide a feasible option for conducting NAT. Reducing the time from specimen collection to submission of specimens for NAT is an important step toward maximizing the public health impact of identifying acute infections.

Stage-specific control of early B cell development by the transcription factor Ikaros.

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell-specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

4th generation HIV screening in Massachusetts: a partnership between laboratory and program.

BACKGROUND: The Massachusetts Department of Public Health's (MDPH) Office of HIV/AIDS (OHA) and Hinton State Laboratory Institute (HSLI) have offered HIV screening since 1985. Point-of-care screening and serum collection for laboratory-based testing is conducted at clinic and non-clinic-based sites across Massachusetts as part of an integrated communicable disease screening intervention. OBJECTIVES AND PROJECT DESIGN: MDPH aimed to transition to a 4th generation HIV screening-based algorithm for testing all serum specimens collected at OHA-funded programs and submitted to the HSLI to detect acute HIV infections, detect and differentiate HIV-1 and HIV-2 infections, eliminate indeterminate results, reduce cost and turnaround time, and link newly diagnosed HIV+ individuals to care. The HSLI and OHA created a joint project management team to plan and lead the transition. RESULTS: The laboratory transitioned successfully to a 4th generation screening assay as part of a revised diagnostic algorithm. In the 12 months since implementation, a total of 7984 serum specimens were tested with 258 (3.2%) positive for HIV-1 and one positive for HIV-2. Eight were reported as acute HIV-1 infections. These individuals were linked to medical care and partner services in a timely manner. Turnaround time was reduced and the laboratory realized an overall cost savings of approximately 15%. CONCLUSIONS: The identification of eight acute HIV infections in the first year underscores the importance of using the most sensitive screening tests available. A multi-disciplinary program and laboratory team was critical to the success of the transition, and the lessons learned may be useful for other jurisdictions.

Essential role of EBF1 in the generation and function of distinct mature B cell types.

The transcription factor EBF1 is essential for lineage specification in early B cell development. In this study, we demonstrate by conditional mutagenesis that EBF1 is required for B cell commitment, pro-B cell development, and subsequent transition to the pre-B cell stage. Later in B cell development, EBF1 was essential for the generation and maintenance of several mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular (FO) and germinal center (GC) B cells were reduced in the absence of EBF1. Activation of the B cell receptor resulted in impaired intracellular signaling, proliferation and survival of EBF1-deficient FO B cells. Immune responses were severely reduced upon Ebf1 inactivation, as GCs were formed but not maintained. ChIP- and RNA-sequencing of FO B cells identified EBF1-activated genes that encode receptors, signal transducers, and transcriptional regulators implicated in B cell signaling. Notably, ectopic expression of EBF1 efficiently induced the development of B-1 cells at the expense of conventional B cells. These gain- and loss-of-function analyses uncovered novel important functions of EBF1 in controlling B cell immunity.

Signs Observed Among Animal Species Infected with Raccoon Rabies Variant Virus, Massachusetts, USA, 1992-2010.

We analyzed signs occurring among domestic and wild terrestrial animal species infected with raccoon rabies variant virus (RRV) in Massachusetts, 1992-2010. The clinical sign of aggression was significantly associated with rabid stray cats (odds ratio, OR = 2.3) and RRV affected major wild terrestrial animal species individually, which included raccoons (OR = 2.8), skunks (OR = 8.0), gray foxes (OR = 21.3), red foxes (OR = 10.4), woodchucks (OR = 4.7) and coyotes (OR = 27.6). While aggression is a useful predictor of rabies among wild animals, combinations of other signs such as ataxia, disorientation, and salivation are useful predictors of rabies among domestic animals. Pets reported with multiple clinical signs had significantly higher rabies positive testing result than those reported with single clinical sign (p < 0.001). The result suggested the importance of avoiding aggressive terrestrial wild animals and giving additional attention to pets with multiple clinical signs.

Increased serum iron levels and infectious complications after liver transplantation.

BACKGROUND: Elevated serum iron levels have been associated with infectious outcomes in various patient populations but, to our knowledge, have never been studied after liver transplantation. METHODS: The relationship between serum iron levels and infectious outcomes after liver transplantation was evaluated in a nested case-control study using prospectively collected data and serum samples. Unadjusted and adjusted hazard ratios were calculated for each iron marker predictor variable (iron level, unsaturated iron-binding capacity, total iron-binding capacity, transferrin saturation, and ferritin level) and time to development of each of 6 outcomes (cytomegalovirus [CMV] disease, invasive fungal infection, bacteremia, invasive fungal infection or bacteremia, any infection, and 1-year mortality rate). RESULTS: Serum measurements (n = 109) corresponding to increased levels of serum iron were independently associated with an increased risk of any infection and death. After adjusting for the number of red blood cell transfusions, donor CMV-seropositive status, and fungal colonization, ferritin level was independently associated with the development of any infection (hazard ratio, 1.09; 95% confidence interval, 1.04-1.14). After adjusting for the number of red blood cell transfusions, development of CMV disease, and administration of intravenous steroids for treatment of rejection, ferritin level was also was independently associated with death (hazard ratio, 1.11; 95% confidence interval, 1.04-1.18). Similar results were found for unsaturated iron binding capacity for the same 2 outcomes. CONCLUSIONS: A better understanding of iron metabolism and its relationship to infection could help guide future infection prognosis, prevention, and management efforts in this high-risk population.