A Phase Ib Trial of AVID200, a TGFß 1/3 Trap, in Patients with Myelofibrosis.

PURPOSE: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by systemic symptoms, cytopenias, organomegaly, and bone marrow fibrosis. JAK2 inhibitors afford symptom and spleen burden reduction but do not alter the disease course and frequently lead to thrombocytopenia. TGFß, a pleiotropic cytokine elaborated by the MF clone, negatively regulates normal hematopoiesis, downregulates antitumor immunity, and promotes bone marrow fibrosis. Our group previously showed that AVID200, a potent and selective TGFß 1/3 trap, reduced TGFß1-induced proliferation of human mesenchymal stromal cells, phosphorylation of SMAD2, and collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PC) with wild-type JAK2 rather than JAK2V617F. PATIENTS AND METHODS: We conducted an investigator-initiated, multicenter, phase Ib trial of AVID200 monotherapy in 21 patients with advanced MF. RESULTS: No dose-limiting toxicity was identified at the three dose levels tested, and grade 3/4 anemia and thrombocytopenia occurred in 28.6% and 19.0% of treated patients, respectively. After six cycles of therapy, two patients attained a clinical benefit by IWG-MRT criteria. Spleen and symptom benefits were observed across treatment cycles. Unlike other MF-directed therapies, increases in platelet counts were noted in 81% of treated patients with three patients achieving normalization. Treatment with AVID200 resulted in potent suppression of plasma TGFß1 levels and pSMAD2 in MF cells. CONCLUSIONS: AVID200 is a well-tolerated, rational, therapeutic agent for the treatment of patients with MF and should be evaluated further in patients with thrombocytopenic MF in combination with agents that target aberrant MF intracellular signaling pathways.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.

BACKGROUND: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. METHODS: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. RESULTS: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. CONCLUSIONS: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions.

BACKGROUND: The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs. METHODS: Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2-/hrp3+), HB3 (hrp2+/hrp3-) and 3BD5 (hrp2-/hrp3-)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH. RESULTS: At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density. CONCLUSIONS: Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

A Multidisciplinary Approach to Implement Personalized Breast Cancer Treatment and Care Plans.

Implementation of survivorship care plans remain a challenge. This quality improvement initiative aims to integrate personalized treatment plans (PTP) and care plans (PCP) into the existing workflow for breast cancer (BC) patients. Methods: Phase 1 was to identify multidisciplinary team members to generate and deliver PTP and PCP. Concurrently, Phase 2 was to deliver PTP and PCP to newly diagnosed invasive BC patients at chemotherapy initiation and completion, respectively. Iterative plan, do, study, act (PDSA) cycles were applied to refine the process. The proportion of information completed for PTP and PCP generation and its delivery by the care team were measured. Patient and provider satisfaction were also assessed. Implementation Process and Results: The care transfer facilitator (CTF) was identified to complete and deliver PTP, and their data entry increased from 0% to 76%, 80%, 92% consecutively during the last 4 PDSA cycles. PTP and PCP were provided to 85% of eligible BC patients. Patients agreed that PTP helped them to actively participate in their care (88%) and communicate with the oncology care team (86%). Primary care physicians agreed that PTP and PCP had the information needed to "stay in the loop" (80%), and oncologists agreed they should be incorporated into oncology clinics (100%). Conclusions: Integrating PTP and PCP generation and delivery into existing workflow has led to an increase in uptake, sustainability and provider buy-in. With limited resources, it remains difficult to find care team members to complete the forms. A dedicated personnel or survivorship clinic is required to successfully implement PTP and PCP as the standard of care.

Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites. METHODS: Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3. RESULTS: RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL. CONCLUSIONS: The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

Web-based Oncology Educational Tool for Medical Trainees on Oncology Rotation-Results of a Pilot Study.

Oncology education for post-graduate medical trainees is mostly clinic-based with didactic lectures. However, a 3-4-week rotation lacks full exposure to the vast field of oncology, resulting in an educational gap. We felt there is a need for a standard curriculum to educate trainees on common oncology topics and encourage self-directed learning. This study aims to improve knowledge of oncology in trainees through the use of an oncology educational tool (consisting of a handbook and website) that we developed and evaluated. Fifty-three post-graduate trainees (years 1, 2, and 3) consented to participate at the start of their oncology rotation. In phase I, four participants took part in a usability evaluation of the tool. In phase II, 39 trainees underwent a knowledge assessment with use of the tool. Baseline and post-intervention test results were compared using paired t tests. In the qualitative study (phase III), 10 trainees provided feedback on the updated tool and overall rotation experience. Issues identified from phase I were addressed prior to subsequent phases. Phase II analysis of complete sets of data found the mean post-intervention scores (9.44/10) were significantly higher (p < 0.001) than the mean baseline scores (7.47/10). In the qualitative study, feedback strongly supported the integration of the tool for improving knowledge of trainees. To our knowledge, this is the first study to show that an oncology educational tool for medical trainees improves oncology knowledge by providing a standard curriculum. Future work involves evaluating this tool to determine if effects are from the education tool or rotation experience.

High-Flow Nasal Oxygen in Patient With Obstructive Sleep Apnea Undergoing Awake Craniotomy: A Case Report.

Patients with obstructive sleep apnea are frequently considered unsuitable candidates for awake craniotomy due to anticipated problems with oxygenation, ventilation, and a potentially difficult airway. At present, only a handful of such accounts exist in the literature. Our report describes the novel use of high-flow nasal oxygen therapy for a patient with moderate obstructive sleep apnea who underwent an awake craniotomy under deep sedation. The intraoperative application of high-flow nasal oxygen therapy achieved satisfactory oxygenation, maintained the partial carbon dioxide pressure within a reasonable range even during periods of deep sedation, permitted responsive patient monitoring during mapping, and provided excellent patient and surgeon satisfaction.

Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Photochemical treatment of blood products could help prevent transfusion-transmitted malaria and reduce the need for donor deferrals. In this study we evaluated the effectiveness of riboflavin and ultraviolet (UV) light against both Plasmodium falciparum, which causes the most severe form of human malaria, and Plasmodium yoelii, an in vivo murine model for malaria. STUDY DESIGN AND METHODS: Plasma and platelet (PLT) concentrates were inoculated with either P. falciparum- or P. yoelii-infected red blood cells (RBCs). Aliquots from each unit were collected after inoculation, after addition of riboflavin, and after treatment. In vitro P. falciparum growth was assessed using thin blood films of duplicate samples at 24, 48, 72, and 96 hours. P. yoelii parasitemia was followed in mice for 14 days postinoculation. RESULTS: In the in vitro studies, the mean P. falciparum parasitemia increased 12- to 19-fold in pretreatment samples, both before and after addition of riboflavin, after 96-hour culture. Few parasites were observed in Mirasol-treated units at 24 hours; those that were observed were degenerating. Through the remainder of the 96-hour culture period, cultures of treated samples were negative. In the in vivo study, mouse plasma containing P. yoelii-infected RBCs had a mean starting titer of 4.6 log mouse infectious dose 50%/mL. No infectious parasite was detected in treated samples. CONCLUSION: Treatment with riboflavin and UV light was effective at reducing viable P. falciparum in both PLT and plasma products by at least 3.2 logs. Additionally, an at least 4.4-log reduction was observed with P. yoelii.