Improved outcomes and cost savings for patients with bleeding disorders: a quality improvement project.

Background: Providing optimal care for patients with bleeding disorders according to national standards remains a challenge at designated Hemophilia Treatment Centers (HTCs). Improved care may reduce bleeds and costs. Objectives: To improve care and demonstrate cost savings by 1) reducing preventable hospitalizations and emergency room visits (PHER) for bleeding, 2) increasing use of prophylaxis in severe hemophilia, and 3) improving patient-HTC communication and primary care engagement. Methods: Prospective quality improvement project using the Define, Measure, Analyze, Improve, and Control methodology to implement uniform guideline-based bleeding disorder care at a rural HTC (N = 88). Intervention used a standardized physician checklist, improved communication, and reserved physician time for urgent management. Outcomes were determined by retrospective chart review; urgent management was tracked prospectively. Results: Intervention significantly reduced PHER by 85.4%. Use of prophylaxis in persons with severe hemophilia increased from 58.8% to 100%; attainment of a primary care physician and electronic portal enrollment met outcomes for intervention success. HTC clinic visit attendance was low at 55.2%. The majority of patients (71.6%) had at least 1 outpatient urgent episode (mean, 0.72 episode per year), and 93% had nonurgent management (mean, 9.3 episodes per year) occurring outside of a clinic visit. Hospital PHER factor cost in the group was reduced by 94.5%, from $11,800 to $640 per patient per year-a cost savings of $982,088 yearly. Conclusion: This collaborative study shows that implementation of a carefully designed quality improvement project, such as uniform guidelines with focus on strengthening ambulatory management, led to improved outcomes and cost savings.

Systems controls are needed to reduce mistaken tests for hemophagocytic lymphohistiocytosis, results of a prospective quality-improvement cohort study.

ABSTRACT: Medical diagnosis and therapy often rely on laboratory testing. We observed mistaken testing in evaluations for hemophagocytic lymphohistiocytosis (HLH) that led to delays and adverse outcomes. Physicians were mistakenly ordering interleukin-2 and quantitative natural killer cell flow cytometry, rather than soluble interleukin 2 receptor (sIL2R) or qualitative natural killer functional tests in the evaluation of patients suspected to have HLH.We initiated a prospective quality improvement project to reduce mistaken testing, reduce delays in correct testing due to mistaken ordering, and improve HLH evaluations. This consisted of provider education, developing an evaluation algorithm, and ultimately required systems interventions such as pop-ups and removal of the mistaken tests from the electronic ordering catalog.Active education reduced mistaken testing significantly in HLH evaluations from baseline (73.3% vs 33.3%, P = .003, relative risk reduction (RRR) 54.5%), but failed to meet the pre-specified RRR cutoff for success (70%). Education alone did not significantly reduce the proportion of HLH evaluations with delays in sIL2R testing (23.3% vs 7.4%, P = .096). Mistaken testing increased after the active intervention ended (33.3% vs 43.5%, P = .390, with RRR 40.7% from baseline. Mistaken test removal was successful: mistaken testing dropped to 0% (P < .001, RRR 100%), saved $14,235 yearly, eliminated delays in sIL2R testing from mistaken testing (23.3% vs 0%, P = .008), and expedited sIL2R testing after admission for HLH symptoms (14.6 days vs 3.8 days, P = .0012). These data show systems controls are highly effective in quality improvement while education has moderate efficacy.

Immune-Related Adverse Events (irAE) in Cancer Immune Checkpoint Inhibitors (ICI) and Survival Outcomes Correlation: To Rechallenge or Not?

INTRODUCTION: There is growing recognition of immune related adverse events (irAEs) from immune checkpoint therapies being correlated with treatment outcomes in certain malignancies. There are currently limited data or consensus to guide management of irAEs with regards to treatment rechallenge. METHODS: We conducted a retrospective analysis with an IRB-approved protocol of adult patients seen at the WVU Cancer Institute between 2011-2019 with a histopathologic diagnosis of active cancers and were treated with immune checkpoint inhibitors (ICI) therapy. RESULTS: Demographics were similar between the ICI interrupted irAE groups within cancer types. Overall, out of 548 patients who received ICI reviewed, there were 133 cases of ≥1 irAE found of any grade. Being treated with anti-CTLA-4 inhibitor ICI was associated with lower risk of death compared to anti-PD-1 ICI. The overall survival difference observed for irAE positive patients, between rechallenged (37.8 months, reinitiated with/without interruption; 38.6 months, reinitiated after interruption) and interrupted/non-reinitiated (i.e., discontinued) groups (24.9 months) was not statistically significant, with a numerical trend favoring the former. CONCLUSIONS: Our exploratory study did not identify significantly different survival outcomes among the Appalachian West Virginia adult cancer patients treated with ICI who developed irAE and had treatment reinitiated after interruption, when compared with those not reinitiated.

A Minimal Parameter Set Facilitating Early Decision-making in the Diagnosis of Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune dysregulation syndrome characterized by uncontrolled immune cell activation. Timely diagnosis is important, since early treatment can improve survival rates. However, completing all assessments needed to reach ≥5 positive criteria out of the 8 HLH-2004 criteria can be time consuming and may delay timely initiation of treatment. Hence, we applied a data-driven approach to identify a minimal parameter set for early decision-making towards the initiation of HLH-specific treatment. We retrospectively evaluated 165 patients from five Dutch tertiary hospitals with suspected HLH. Sixteen pHLH (median age 0.5 years) and 70 sHLH patients (median age 8.7 years) were identified using the HLH-2004 criteria. Clustering analysis and multi-receiver operator characteristics were used to identify parameters distinctive of HLH. The presence of either increased ferritin, cytopenia in ≥2 lineages, or splenomegaly distinguished HLH from non-HLH cases with a negative predictive value of 100%. A minimal parameter set consisting of 2 major criteria (phagocytosis and splenomegaly) and 3 minor criteria (cytopenia, increased ferritin, and increased triglycerides/low fibrinogen) predicted HLH with 95% (88-99) sensitivity and 94% (86-98) specificity. This finding was replicated in an independent retrospective validation cohort of 109 US patients (n = 109). By dividing a subset of the HLH-2004 criteria into major and minor criteria, this strategy uses the evaluation of less than 5 criteria to quickly identify patients with HLH. When confirmed in a prospective setting, this approach could be of value for timely diagnosis and treatment of HLH.

Properdin Is a Key Player in Lysis of Red Blood Cells and Complement Activation on Endothelial Cells in Hemolytic Anemias Caused by Complement Dysregulation.

The complement system alternative pathway (AP) can be activated excessively in inflammatory diseases, particularly when there is defective complement regulation. For instance, deficiency in complement regulators CD55 and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), whereas Factor H mutations predispose to atypical hemolytic uremic syndrome (aHUS), both causing severe thrombohemolysis. Despite eculizumab being the treatment for these diseases, benefits vary considerably among patients. Understanding the molecular mechanisms involved in complement regulation is essential for developing new treatments. Properdin, the positive AP regulator, is essential for complement amplification by stabilizing enzymatic convertases. In this study, the role of properdin in red blood cell (RBC) lysis and endothelial cell opsonization in these AP-mediated diseases was addressed by developing in vitro assays using PNH patient RBCs and human primary endothelial cells, where the effects of inhibiting properdin, using novel monoclonal antibodies (MoAbs) that we generated and characterized, were compared to other complement inhibitors. In in vitro models of PNH, properdin inhibition prevented hemolysis of patient PNH type II and III RBCs more than inhibition of Factor B, C3, and C5 (>17-fold, or >81-fold, or >12-fold lower molar IC90 values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC90 values than inhibition of Factor B, or C3, respectively (P < 0.0001). When comparing target/inhibitor ratios in all hemolysis assays, inhibiting properdin was at least as efficient as the other complement inhibitors in most cases. In addition, using in vitro endothelial cell assays, the data indicate a critical novel role for properdin in promoting complement activation on human endothelial cells exposed to heme (a hemolysis by-product) and rH19-20 (to inhibit Factor H cell-surface protection), as occurs in aHUS. Inhibition of properdin or C3 in this system significantly reduced C3 fragment deposition by 75%. Altogether, the data indicate properdin is key in promoting RBC lysis and complement activation on human endothelial cells, contributing to the understanding of PNH and aHUS pathogenesis. Further studies to determine therapeutic values of inhibiting properdin in complement-mediated diseases, in particular those that are characterized by AP dysregulation, are warranted.

Ruxolitinib in adult patients with secondary haemophagocytic lymphohistiocytosis: an open-label, single-centre, pilot trial.

BACKGROUND: Hemophagocytic lymphohistiocytosis is a cytokine-driven inflammatory syndrome that is associated with substantial morbidity and mortality. Overall survival in adult patients with secondary haemophagocytic lymphohistiocytosis remains suboptimal, and novel therapeutic strategies are needed. The phosphorylation-dependent activation of the Janus family kinases JAK1 and JAK2 are hallmarks of the final common pathway in this disease. We therefore aimed to determine the activity and safety of ruxolitinib, a JAK inhibitor, in adults with secondary haemophagocytic lymphohistiocytosis. METHODS: We performed an open-label, single-centre, pilot study of ruxolitinib in adults with secondary haemophagocytic lymphohistiocytosis at the University of Michigan Rogel Cancer Center (Ann Arbor, MI, USA). We included patients aged 18 years or more who fulfilled at least five of the eight HLH-2004 criteria for hemophagocytic lymphohistiocytosis. Discontinuation of corticosteroids was not required for enrolment in this study. Patients received oral ruxolitinib (15 mg twice a day) on a continuous 28-day cycle, or until disease progression or unacceptable toxicity. The primary endpoint was overall survival at 2 months from the first dose of ruxolitinib. Secondary endpoints included the assessment of adverse events, response (defined as the assessment of all quantifiable signs and laboratory abnormalities included in the diagnostic criteria for haemophagocytic lymphohistiocytosis), and pharmacodynamic biomarkers. Analyses were done in all treated patients with available data. This study is registered with ClinicalTrials.gov, number NCT02400463, and is still recruiting. FINDINGS: As of Feb 7, 2019, five patients had been enrolled. The first patient was enrolled in February, 2016. No deaths were recorded, with a median follow-up of 490 days (IQR 190-1075). 2-month overall survival was 100% (95% CI 57-100). Regarding response, resolution of symptoms (either partial or complete) and disease-associated laboratory abnormalities was observed in all five patients. Cytopenias improved in all patients within the first week of treatment, leading to relatively rapid transfusion independence, discontinuation of corticosteroids, and hospital discharge. A single serious adverse event (ie, grade 4 febrile neutropenia) was reported. One patient discontinued treatment because of grade 2 extremity pain and no treatment-related deaths were observed. Improvements in inflammatory markers (eg, ferritin, soluble IL-2 receptor) and T cells and monocytes activation (ie, decreased STAT1 phosphorylation) were observed following treatment. INTERPRETATION: These preliminary data suggest that ruxolitinib is active, well tolerated, and manageable in the outpatient setting in patients with secondary haemophagocytic lymphohistiocytosis. Given the paucity of effective, non-myelosuppressive therapies, these preliminary findings have important therapeutic implications for patients with haemophagocytic lymphohistiocytosis and other cytokine-release syndromes and warrant further investigation. FUNDING: National Cancer Institute, the University of Michigan Rogel Cancer Center, and Incyte Corporation.

A case-control analysis of hyperhemolysis syndrome in adults and laboratory correlates of complement involvement.

BACKGROUND: Hyperhemolysis syndrome (HS) is a poorly understood, severe hemolytic anemia provoked by transfusion. Both host and donor RBCs are destroyed in HS; thus, transfusion paradoxically worsens anemia. Risk factors and mechanism of HS are unknown. STUDY DESIGN AND METHODS: A retrospective case-control analysis was performed on adults with HS. Patients with HS were matched 1:1 with matched, transfused controls, and HS risk factors were analyzed with multivariable logistic regression. HS samples were analyzed for complement deposition by flow cytometry, and an in vitro model of bystander hemolysis was developed. RESULTS: Forty-one patients with 54 episodes of HS were identified in a 26-year period from 1992 to 2018. Of the HS episodes, only 18.5% were associated with a new alloantibody, and such patients were more tolerant of additional transfusion in the acute episode (p = 0.005). Thirteen percent of episodes were fatal, and HS recurred in 52.6%. Alloimmunization (odds ratio [OR], 17.3), non-B blood type (OR, 9.8), D antigen (OR, 9.1), and infection (OR, 5.5) were associated with HS on multivariable analysis. Hyperbilirubinemia was predictive of fatal HS (OR, 33.6). Increased complement was observed on RBCs during HS episodes, and the in vitro model of bystander hemolysis recapitulated complement decoration of sickled RBCs. CONCLUSIONS: HS is associated with significant morbidity, mortality, and recurrence. Risk factors such as known alloimmunization, blood group, and infection predispose to HS. Bystander complement activation may drive HS. These factors may help physicians refine risk-benefit assessments for transfusion and guide further therapeutic development.

Complement-driven anemia: more than just paroxysmal nocturnal hemoglobinuria.

Atypical hemolytic uremic syndrome (aHUS); hemolysis, elevated liver function tests, and low platelets syndrome; and transplant-associated thrombotic microangiopathy are related conditions, in that many patients harbor germline heterozygous mutations in genes that regulate the alternative pathway of complement (APC). Penetrance is variable because development of clinically significant disease appears to require supervention of a process such as inflammation. Complement activation on the endothelial surfaces leads to endothelial damage, platelet consumption, microthrombi, and a mechanical hemolytic anemia with schistocytes. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic disease caused by expansion of a stem cell that harbors a somatic mutation in PIGA PIGA mutant blood cells are deficient in the complement regulator proteins CD55 and CD59, making them susceptible to intravascular hemolysis due to a failure to regulate the APC on erythrocytes. Eculizumab is a monoclonal antibody that binds to C5 and inhibits terminal complement by interfering with the cleavage of C5 by the C5 convertases. The drug is approved by the US Food and Drug Administration for the treatment of aHUS and PNH; however, a new generation of complement inhibitors that block C5 and other components of the complement cascade is showing promise in preclinical and clinical trials.

A prospective quality improvement initiative in adult hemophagocytic lymphohistiocytosis to improve testing and a framework to facilitate trigger identification and mitigate hemorrhage from retrospective analysis.

Hemophagocytic lymphohistiocytosis (HLH) is a highly fatal, hyperinflammatory syndrome in adults triggered by an underlying illness in most cases. As such, suspicion of HLH dictates further investigation to identify the HLH trigger and determine treatment. HLH is clinically challenging due to diverse presentations and underlying triggers, provider unfamiliarity, and bleeding complications. Clinically, we observed diagnostic error from incorrect testing and cognitive biases (interleukin-2 confused with soluble interleukin-2 receptor and natural killer cell quantification confused with functional assays).This study reports our single institutional experience with adult HLH with the aim to reduce erroneous testing with a quality improvement (QI) project, and to facilitate trigger discovery and mitigate hemorrhage. Provider education on HLH testing was the prospective intervention, followed by mistaken test removal. HLH triggers and diagnostic utility were determined by retrospective chart review. Risk factors for hemorrhage were determined by multivariable analysis.Erroneous HLH testing was reduced from 74% to 24% of patients (P < .001) by the QI intervention. These changes were projected to save $11,700 yearly. The majority (64%) of patients evaluated for HLH were on non-hematology/oncology services, highlighting the need for vigilance in hematology consultation. Sixty-three patients met classic HLH-2004 criteria for HLH. Malignancy (38%), infection (27%), Epstein-Barr virus (EBV) (14%), or autoimmune disease (8%) triggered most HLH cases. HLH triggers were most commonly identified by serologic testing (27%) and bone marrow biopsy (19%). Biopsy of other affected organs based on PET-CT imaging after unsuccessful initial diagnostic measures was helpful, and focal fluorodeoxyglucose uptake was predictive of an underlying malignancy (likelihood ratio 8.3, P = .004). Major hemorrhage occurred in 41% of patients. On multivariable analysis the odds ratios (OR) for major hemorrhage were increased for patients with intensive care unit level care (OR 10.47, P = .005), and disseminated intravascular coagulation in the first week of admission (OR 10.53, P = .04).These data are incorporated into a framework to encourage early HLH recognition with the HScore, facilitate trigger identification, identify those at risk for hemorrhage, and minimize low-yield or erroneous testing.

Germline mutations in the alternative pathway of complement predispose to HELLP syndrome.

BACKGROUND: HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome is a severe variant of hypertensive disorders of pregnancy affecting approximately 1% of all pregnancies, and has significant maternal and fetal morbidity. Previously, we showed that upregulation of the alternative pathway of complement (APC) plays a role in HELLP syndrome. We hypothesize that HELLP syndrome follows a 2-hit disease model similar to atypical hemolytic uremic syndrome (aHUS), requiring both genetic susceptibility and an environmental risk factor. Our objective was to perform a comparative analysis of the frequency of APC activation and germline mutations in affected women and to create a predictive model for identifying HELLP syndrome. METHODS: Pregnant women with HELLP syndrome, and healthy controls after 23 weeks of gestation were recruited, along with aHUS and thrombotic thrombocytopenic purpura participants. We performed a functional assay, the mHam, and targeted genetic sequencing in all groups. RESULTS: Significantly more participants with rare germline mutations in APC genes were present in the HELLP cohort compared with controls (46% versus 8%, P = 0.01). In addition, significantly more HELLP participants were positive for the mHam when compared with controls (62% versus 16%, P = 0.009). Testing positive for both a germline mutation and the mHam was highly predictive for the diagnosis of HELLP syndrome. CONCLUSION: HELLP syndrome is characterized by both activation of the APC and frequent germline mutations in APC genes. Similar to aHUS, treatment via complement inhibition to mitigate maternal and fetal morbidity and mortality may be possible. FUNDING: National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.

Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition.

VPS4 proteins are AAA(+) ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA(+) ATPases, but this has been difficult to directly demonstrate. We found that concentrating His(6)-VPS4A on liposomes containing Ni(2+)-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.

Cell-free reconstitution of multivesicular body formation and receptor sorting.

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.

Cell biology of the ESCRT machinery.

The ESCRT (endosomal sorting complex required for transport) machinery comprises a set of protein complexes that regulate sorting and trafficking into multivesicular bodies en route to the lysosome. The physical mechanism responsible for generating lumenal vesicles in this pathway is unknown. Here we review recent studies suggesting that components of the ESCRT-III complex drive lumenal vesicle formation and consider possible mechanisms for this reaction.

Novel interactions of ESCRT-III with LIP5 and VPS4 and their implications for ESCRT-III disassembly.

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

Phylogeography and population structure of Aedes aegypti in Arizona.

Aedes aegypti, the mosquito responsible for transmitting dengue, has colonized many cities and towns throughout Arizona. Determining both the migration between, and the origin of, local Ae. aegypti populations is important for vector control and disease prevention purposes. Amplified fragment length polymorphism was used to infer geographic structure and local substructure, and effective migration rates (M, migrants per generation) between populations, and to determine genetic differentiation between populations (PhiPT). Three geographically and genetically differentiated groups of populations were identified. Population substructure was only detected in the border town of Nogales. Reliable estimates of M between regions ranged from 1.02 to 3.41 and between cities within regions from 1.66 to 4.44. In general, pairwise PhiPT were lowest between cities within regions. The observed patterns of genetic differentiation suggest infrequent migration between populations and are compatible with the idea of human transport facilitating dispersal between regions.

Population and parity levels of Aedes aegypti collected in Tucson.

Oviposition traps were used to follow changes in the population of Aedes aegypti (L.) (Diptera:Culicidae) in a seven-block area in midtown region of Tucson, Arizona. About 20,000 eggs were collected over a period from 1 June to 14 October 2000. Peak mosquito populations were correlated with the late summer rains. Mosquitoes seeking a blood meal were collected and dissected to determine if they had previously fed, i.e. if they were parous. Of the 241 females examined, 44% were parous, with a range from 0% to 80%. Females that had blood in their guts were collected and the source of blood was identified using an ELISA. Preliminary results suggest that 80% of them had fed on humans. These data suggest that the reproductive history of Tucson populations of Ae. aegypti could be conducive for transmission of dengue viruses.