The ECHELON-2 Trial: 5-year results of a randomized, phase III study of brentuximab vedotin with chemotherapy for CD30-positive peripheral T-cell lymphoma.

BACKGROUND: For patients with peripheral T-cell lymphoma (PTCL), outcomes using frontline treatment with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like therapy are typically poor. The ECHELON-2 study demonstrated that brentuximab vedotin plus cyclophosphamide, doxorubicin, and prednisone (A+CHP) exhibited statistically superior progression-free survival (PFS) per independent central review and improvements in overall survival versus CHOP for the frontline treatment of patients with systemic anaplastic large cell lymphoma or other CD30-positive PTCL. PATIENTS AND METHODS: ECHELON-2 is a double-blind, double-dummy, randomized, placebo-controlled, active-comparator phase III study. We present an exploratory update of the ECHELON-2 study, including an analysis of 5-year PFS per investigator in the intent-to-treat analysis group. RESULTS: A total of 452 patients were randomized (1 : 1) to six or eight cycles of A+CHP (N = 226) or CHOP (N = 226). At median follow-up of 47.6 months, 5-year PFS rates were 51.4% [95% confidence interval (CI): 42.8% to 59.4%] with A+CHP versus 43.0% (95% CI: 35.8% to 50.0%) with CHOP (hazard ratio = 0.70; 95% CI: 0.53-0.91), and 5-year overall survival (OS) rates were 70.1% (95% CI: 63.3% to 75.9%) with A+CHP versus 61.0% (95% CI: 54.0% to 67.3%) with CHOP (hazard ratio = 0.72; 95% CI: 0.53-0.99). Both PFS and OS were generally consistent across key subgroups. Peripheral neuropathy was resolved or improved in 72% (84/117) of patients in the A+CHP arm and 78% (97/124) in the CHOP arm. Among patients who relapsed and subsequently received brentuximab vedotin, the objective response rate was 59% with brentuximab vedotin retreatment after A+CHP and 50% with subsequent brentuximab vedotin after CHOP. CONCLUSIONS: In this 5-year update of ECHELON-2, frontline treatment of patients with PTCL with A+CHP continues to provide clinically meaningful improvement in PFS and OS versus CHOP, with a manageable safety profile, including continued resolution or improvement of peripheral neuropathy.

Unusual Stability of a Recombinant Verrucomicrobium spinosum Tyrosinase to Denaturing Agents and Its Use for a Production of a Protein with Adhesive Properties.

Tyrosinases catalyze oxidation of phenols with a formation of biphenols, quinones, and highly polymerized melanins. Tyrosinases have prospects for industrial use to remove phenols, also in biosensors, in bioorganic synthesis, and for a production of biocompatible adhesives (medical glues). Despite growing fields of potential applications, a selection of commercially available tyrosinases are currently limited to a single enzyme which is isolated from fruiting bodies of mushrooms. This article describes a preparation of recombinant tyrosinase from a bacterium Verrucomicrobium spinosum using a heterologous expression in Escherichia coli. Recombinant V. spinosum tyrosinase has high specific activity (13,200 U/mg). A resistance of the enzyme was investigated to chemical agents used to denature proteins and keep poorly solvable proteins in a solution. The enzyme preserves activity in the presence of urea and retains at least a fraction of its enzymatic activity at concentrations of urea up to 4.5 M. An addition of sodium lauroyl sarcosinate to 1 or 2% activates the tyrosinase. Novel means of quantitatively expressing tyrosinase activity is described in this article. The method uses a set of parameters obtained from non-linear estimation of the progress curves and is suitable for enzymatic reactions which do not comply with Michaelis-Menten kinetics. Tyrosinase may be used to introduce into proteins a post-translational modification which is a conversion of tyrosine residues (Tyr) into residues of 3,4-dioxyphenylalanine (DOPA). The presence of DOPA provides the polypeptides with a capability of strong molecular adhesion. Co-expression of tyrosinase and a recombinant protein mimicking marine mussel-encoded adhesive proteins resulted in obtaining of the protein in which at least a part of Tyr residues had been converted to DOPA. The DOPA-containing protein had high adhesion strength (2.5 MPa).

Phase II study of idelalisib, a selective inhibitor of PI3Kδ, for relapsed/refractory classical Hodgkin lymphoma.

BACKGROUND: The phosphatidylinositol-3-kinase delta (PI3Kδ) inhibitor idelalisib has been shown to block downstream intracellular signaling, reduce the production of prosurvival chemokines and induce apoptosis in classical Hodgkin lymphoma (HL) cell lines. It has also been shown to inhibit regulatory T cells and myeloid-derived suppressor cells in other tumor models. We hypothesized that inhibiting PI3Kδ would have both direct and indirect antitumor effects by directly targeting the malignant cells as well as modulating the inflammatory microenvironment. We tested this hypothesis in a phase II study. PATIENTS AND METHODS: We enrolled 25 patients with relapsed/refractory HL with a median age of 42 years and who had previously received a median of five therapies including 18 (72%) with failed autologous stem cell transplant, 23 (92%) with failed brentuximab vedotin, and 11 (44%) with prior radiation therapy. Idelalisib was administered at 150 mg two times daily; an increase to 300 mg two times daily was permitted at the time of disease progression. RESULTS: The overall response rate to idelalisib therapy was 20% (95% confidence interval: 6.8%, 40.7%) with a median time to response of 2.0 months. Seventeen patients (68%) experienced reduction in target lesions with one complete remission and four partial remissions. The median duration of response was 8.4 months and median progression-free survival was 2.3 months. The most common grade ≥3 adverse event was elevation of alanine aminotransferase (two patients, 8%). Diarrhea/colitis was seen in three patients and was grade 1-2. There was one adverse event leading to death (hypoxia). CONCLUSIONS: Idelalisib was tolerable and had modest single-agent activity in heavily pretreated patients with HL. Rational combinations with other novel agents may improve response rate and duration of response. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov # NCT01393106.

Maintenance rituximab after autologous stem cell transplantation in patients with mantle cell lymphoma.

BACKGROUND: High-dose therapy and autologous stem cell transplantation (ASCT) improves outcomes for patients with mantle cell lymphoma (MCL), but relapse ultimately occurs in most patients. Recently presented interim results from a phase III prospective trial suggest maintenance rituximab (MR) after ASCT for MCL improves progression-free survival (PFS). The maturation of these data and any benefit of MR on overall survival (OS) remain to be defined. PATIENTS AND METHODS: In this retrospective study, we examined a cohort of consecutive patients with MCL that underwent ASCT for MCL at our center and evaluated their outcomes according to whether they received MR after ASCT (n = 50) or did not (n = 107). MR was treated as a time-dependent covariate to account for variation in timing of its initiation. RESULTS: MR was associated with an improved PFS [hazard ratio (HR) 0.44; confidence interval (CI) (0.24-0.80), P = 0.007] and overall survival (OS; HR 0.46; CI 0.23-0.93, P = 0.03) following a multivariate adjustment for confounding factors with a median follow-up of ∼5 years. Grade 4 neutropenia was increased (34% versus 18%, P = 0.04) in the MR group, but no effect on the rate of mortality unrelated to relapse was observed. CONCLUSIONS: These data support that MR after ASCT for MCL confers a benefit in PFS and additionally suggest it may improve OS. General application of this strategy will require confirmation of benefit in prospective randomized trials.

SplitCore Technology Allows Efficient Production of Virus-Like Particles Presenting a Receptor-Contacting Epitope of Human IgE.

Immunoglobulin E (IgE) plays a central role in type I hypersensitivity including allergy and asthma. Novel treatment strategy envisages development of a therapeutic vaccine designed to elicit autologous blocking antibodies against the IgE. We sought to develop an IgE-epitope antigen that induces antibodies against a receptor-contacting epitope on human IgE molecule. We designed the VLP immunogens which utilize hepatitis B virus core protein (HBcAg) as a carrier, and present arrays of the receptor-contacting epitopes of the human IgE on their surfaces. FG loop from the IgE domain Cε3 was engineered into the HBcAg. Two constructs explore a well-established approach of insertion into a main immunodominant region of the HBcAg. Third construct is different in that the carrier is produced in a form of an assembly of two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit (SplitCore technology). No VLPs were isolated from E.coli expressing the IgE-epitope antigens with contiguous sequences. On the contrary, the SplitCore antigen carrying the FG loop efficiently formed the VLPs. Immunization of mice with the VLPs presenting receptor-contacting epitope of the IgE elicited antibodies recognizing the human IgE in ELISA.

Utility of ¹8fluoro-deoxyglucose positron emission tomography for prognosis and response assessments in a phase 2 study of romidepsin in patients with relapsed or refractory peripheral T-cell lymphoma.

BACKGROUND: For patients with peripheral T-cell lymphoma (PTCL), the value of (18)fluoro-deoxyglucose positron emission tomography (FDG-PET) scans for assessing prognosis and response to treatment remains unclear. The utility of FDG-PET, in addition to conventional radiology, was examined as a planned exploratory end point in the pivotal phase 2 trial of romidepsin for the treatment of relapsed/refractory PTCL. PATIENTS AND METHODS: Patients received romidepsin at a dose of 14 mg/m(2) on days 1, 8, and 15 of 28-day cycles. The primary end point was the rate of confirmed/unconfirmed complete response (CR/CRu) as assessed by International Workshop Criteria (IWC) using conventional radiology. For the exploratory PET end point, patients with at least baseline FDG-PET scans were assessed by IWC + PET criteria. RESULTS: Of 130 patients, 110 had baseline FDG-PET scans, and 105 were PET positive at baseline. The use of IWC + PET criteria increased the objective response rate to 30% compared with 26% by conventional radiology. Durations of response were well differentiated by both conventional radiology response criteria [CR/CRu versus partial response (PR), P = 0.0001] and PET status (negative versus positive, P < 0.0001). Patients who achieved CR/CRu had prolonged progression-free survival (PFS, median 25.9 months) compared with other response groups (P = 0.0007). Patients who achieved PR or stable disease (SD) had similar PFS (median 7.2 and 6.3 months, respectively, P = 0.6427). When grouping PR and SD patients by PET status, patients with PET-negative versus PET-positive disease had a median PFS of 18.2 versus 7.1 months (P = 0.0923). CONCLUSIONS: Routine use of FDG-PET does not obviate conventional staging, but may aid in determining prognosis and refine response assessments for patients with PTCL, particularly for those who do not achieve CR/CRu by conventional staging. The optimal way to incorporate FDG-PET scans for patients with PTCL remains to be determined. TRIAL REGISTRATION: NCT00426764.

The Opisthorchis felineus paramyosin: cDNA sequence and characterization of its recombinant fragment.

The cDNA sequence of Opisthorchis felineus paramyosin (PM) was determined and shown to have 66-70% homology with two schistosomes and two cestodes. Phylogenetic analysis revealed an almost equal distance between O. felineus and these distinct clades. Because of its relatively low conservation, the PM gene may be a convenient genetic marker for studying phylogenetic relationships among platyhelminthes.A 25-kDa recombinant polypeptide corresponding to the central part of the full-length PM was produced. In Western blot analysis, murine hyperimmune serum against recombinant PM (recPM) detected 100-kDa polypeptides in the O. felineusegg and somatic antigens. Interactions of recPM with polyclonal anti-parasite antibodies and anti-recPM sera in ELISA with native antigens demonstrated that recPM carries a B cell epitope identical to the O. felineusnative antigen. Our sequence and immunologic data may be helpful in developing new diagnostic tools and candidate vaccines for O. felineus infection.

In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.

Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity.

To determine the role of perforin-mediated cytotoxic T lymphocyte (CTL) effector function in immune regulation, we studied a well-characterized mouse model of graft-versus-host disease (GVHD). Induction of acute GVHD using perforin-deficient donor T cells (pfp-->F1) initially resulted in features of acute GVHD, e.g., engraftment of both donor CD4(+) and CD8(+) T cells, upregulation of Fas and FasL, production of antihost CTL, and secretion of both Th1 and Th2 cytokines. Despite fully functional FasL activity, pfp donor cells failed to totally eliminate host B cells, and, by 4 weeks of disease, cytokine production in pfp-->F1 mice had polarized to a Th2 response. Pfp-->F1 mice eventually developed features of chronic GVHD, such as increased numbers of B cells, persistence of donor CD4 T cells, autoantibody production, and lupuslike renal disease. We conclude that in the setting of B- and T-cell activation, perforin plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and ag-specific T cells. Moreover, an ineffective initial CTL response can evolve into a persistent antibody-mediated response and, with it, the potential for sustained humoral autoimmunity.

Differential expression of Fas and Fas ligand in acute and chronic graft-versus-host disease: up-regulation of Fas and Fas ligand requires CD8+ T cell activation and IFN-gamma production.

The parent-into-F1 model of acute and chronic graft-vs-host disease (GVHD) was used as an example of in vivo cell-mediated or Ab-mediated responses, respectively, and the roles of Fas and Fas ligand (FasL) were investigated. Using both flow cytometry and PCR methodologies, we found that acute GVHD mice exhibited significant up-regulation of Fas and FasL, whereas Fas/FasL up-regulation in chronic GVHD mice was equal to or marginally greater than that in uninjected mice. Functional studies confirmed that Fas/FasL contributed to the anti-host CTL activity of splenocytes from acute GVHD mice, although a perforin-dependent pathway was also identified. Despite the presence of FasL on both donor CD4+ and CD8+ T cells in acute GVHD mice, depletion studies demonstrated that all the in vitro anti-host CTL activity resided in the CD8+ population. Furthermore, injection of CD8-depleted B6 spleen cells into F1 mice blocked Fas/FasL up-regulation and IFN-gamma production, resulting in chronic GVHD. Lastly, up-regulation of Fas/FasL in acute GVHD mice could be blocked by anti-IFN-gamma mAb in vivo. Thus, in this in vivo model of alloantigen immune responsiveness, Fas/FasL up-regulation is critically dependent on Ag-specific (donor) CD8+ T cell activation and IFN-gamma production. Donor CD4+ T cell activation in the absence of CD8+ T cell activation results in an autoantibody-mediated response, no significant Fas/FasL up-regulation, impaired elimination of autoreactive B cells, and persistent humoral autoimmunity.

Heterogeneity of platelet aggregation and major surface receptor expression in patients with acute myocardial infarction.

BACKGROUND: Platelets play an important role in the natural history of acute myocardial infarction (AMI). METHODS AND RESULTS: Platelet aggregation and receptor expression were studied in 23 patients with AMI before reperfusion therapy and compared with 10 healthy control subjects. Platelet aggregation was induced with 5 micromol/L adenosine 5'-diphosphate, 10 micromol/L ADP, 1 microg/mL collagen, 1 mg/mL thrombin, and 1.25 mg/mL ristocetin. Receptor expression was measured by flow cytometry with monoclonal antibodies to p24 (CD9), Ib (CD42b), IIb (CD41b), IIIa (CD61), IIb/IIIa (CD41b/CD61), very late antigen-2 (CD49b), P-selectin (CD62p), platelet/endothelial cell adhesion molecule-1 (CD31); and vitronectin (CD51/CD61). The percentage of platelet aggregation was higher in patients with AMI when induced by 5 micromol/L ADP (64.1+/-12.7 vs 52.0+/-6.7; P=.04), by 10 micromol/L ADP (71.7+/-13.0 vs 59.2+/-7.2, P=.003), by thrombin (75.8+/-10.9 vs 60.5+/-6.9, P=.01), and by ristocetin (92.5+/-7.8 vs 71.3+/-7.4, P=.0001). Collagen-induced platelet aggregation did not differ between groups. Expression of P-selectin (log amplification of fluorescence intensity) (31.5+/-5.0 vs 25.1+/-2.6, P=.003) and platelet/endothelial cell adhesion molecule-1 (56.8+/-17.7 vs 44.5+/-3.7, P=.04) were significantly increased in patients with AMI. The expression of IIb (28.4+/-2.5 vs 37.2+/-1.7, P=.0001) and Ib (103.6+/-29.9 vs 133.8+/-8.0, P=.007) were reduced in patients with AMI. CONCLUSIONS: Platelets are not necessarily systemically activated during the prereperfusion phase of AMI. For each agonist used and surface antigen measured, there was a cohort of patients with AMI within the normal or even below normal range of platelet status.

Effects of reteplase and alteplase on platelet aggregation and major receptor expression during the first 24 hours of acute myocardial infarction treatment. GUSTO-III Investigators. Global Use of Strategies to Open Occluded Coronary Arteries.

OBJECTIVES: We sought to compare platelet characteristics after reteplase and alteplase therapy in the setting of the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO)-III trial. BACKGROUND: Platelet function may be impaired during thrombolysis in patients with an acute myocardial infarction. The effects of reteplase and alteplase on platelet aggregation and major surface antigen expression during the first 24 h of infarction therapy are unknown. METHODS: Platelet aggregation and receptor expression by flow cytometry were determined in 23 patients before thrombolysis and thereafter at 3, 6, 12 and 24 h. RESULTS: Aggregation was higher after reteplase at 24 h when induced by 5 micromol/liter adenosine diphosphate (ADP) (p = 0.007), 10 micromol/liter ADP (p = 0.02), collagen (p = 0.003) and thrombin (p = 0.009) than after alteplase. Reteplase therapy exhibited greater glycoprotein (GP) IIb/IIIa (p = 0.04), very late antigen-2 (p = 0.04) and platelet/endothelial cell adhesion molecule-I (p = 0.002) expression at 24 h. Trends toward decreased receptor expression early (3 to 6 h), followed by a progressive increase at 12 h and especially at 24 h occurred after both agents. CONCLUSIONS: In this prospective clinical ex vivo platelet study, similar patterns of platelet aggregation and surface receptor expression occurred during the first 24 h of coronary thrombolysis with reteplase and alteplase. However, after reteplase, indicators of platelet activity were higher at 24 h after thrombolysis than after alteplase. These data suggest that GP IIb/IIIa inhibitors or other antiplatelet strategies may be particularly advantageous when used 12 to 24 h after thrombolysis, especially after reteplase therapy. It is at this time point during the first day of coronary thrombolysis that GP IIb/IIIa is markedly expressed and platelets are most active.

Depressed platelet status in an elderly patient with hemorrhagic stroke after thrombolysis for acute myocardial infarction. GUSTO-III Investigators.

BACKGROUND: Impaired platelet function has been reported in acute myocardial infarction (AMI) and stroke. However, prospective data on the changes of platelet status in patients before the occurrence of hemorrhagic stroke after thrombolytic therapy are unavailable. CASE DESCRIPTION: An 86-year-old male patient was among the 23 AMI patients enrolled in the platelet study for the GUSTO-III trial. He received 325 mg of aspirin daily for at least 6 years, suffered an AMI, and was successfully reperfused with alteplase, but after 44 hours developed a large hemorrhagic stroke resulting in paraplegia. Platelet aggregation and receptor expression were measured by flow cytometry and ELISA before thrombolysis and at 3, 6, 12, and 24 hours thereafter. The percentage of platelet aggregation was lower in the stroke patient at every time point when induced by 5 micromol/L of ADP, by 10 micromol/L of ADP, and by thrombin than in the rest of the AMI group. Ristocetin and collagen-induced aggregability were within the group range. Decreased platelet glycoprotein Ib, IIb, IIIa, and IIb/IIIa and vitronectin receptor expression were observed in the stroke patient. No other differences in p24 (CD9), very late antigen-2, P-selectin, and platelet/endothelial cell adhesion molecule-1 expression were determined. CONCLUSIONS: Profound depression of platelet status preceded the occurrence of hemorrhagic stroke in an elderly long-term aspirin user treated with thrombolytic therapy. Initial "exhausted" platelets may be responsible for the increased risk for hemorrhagic stroke after coronary thrombolysis.

A major role for the Fas pathway in acute graft-versus-host disease.

Recent studies have suggested a role for the Fas pathway in the wasting syndrome associated with lpr-->wild-type bone marrow transplants. To directly examine whether Fas ligand has a major role in the development of acute graft-vs-host disease (GVHD), Fas ligand-deficient (gld) mice were used as donors and C3H/HeJ x C57BL/6F1 as recipients in the parent-into-F1 model of acute GVHD. Transplantation of C3H/gld spleen cells induced significantly less host lymphoid depletion and was associated with less antihost cytotoxic activity in vitro when compared with wild-type C3H donor cells. The reduced depletion of host lymphocytes was explained by both impaired antihost T cell cytolytic activity and by reduced expansion of gld donor T cells in F1 recipients. These findings not only indicate that the Fas ligand is an important effector molecule in acute GVHD, but also provide in vivo evidence supporting a role for Fas/Fas ligand interactions in T cell expansion and maturation.