Pembrolizumab for advanced prostate adenocarcinoma: findings of the KEYNOTE-028 study.

Background: Patients with castration-resistant prostate cancer derive only modest clinical benefit from available therapies. Blockade of the inhibitory programmed death 1 (PD-1) receptor by monoclonal antibodies has been effective in several malignancies. Results from the prostate adenocarcinoma cohort of the nonrandomized phase Ib KEYNOTE-028 trial of pembrolizumab in advanced solid tumors are presented. Materials and methods: Key eligibility criteria included advanced prostate adenocarcinoma, unsuccessful standard therapy, measurable disease per Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1), and PD-1 ligand (PD-L1) expression in ≥1% of tumor or stromal cells. Patients received pembrolizumab 10 mg/kg every 2 weeks until disease progression or intolerable toxicity for up to 24 months. Primary end point was objective response rate (ORR) per RECIST v1.1 by investigator review. Results: Median patient age in this cohort (n = 23) was 65 years; 73.9% of patients received at least two prior therapies for metastatic disease. There were four confirmed partial responses, for an ORR of 17.4% [95% confidence interval (CI) 5.0%-38.8%]; 8 of 23 (34.8%) patients had stable disease. Median duration of response was 13.5 months. Median progression-free survival (PFS) and overall survival (OS) were 3.5 and 7.9 months, respectively; 6-month PFS and OS rates were 34.8% and 73.4%, respectively. One patient remained on treatment at data cutoff. After a median follow-up of 7.9 months, 14 (60.9%) patients experienced treatment-related adverse events (TRAEs), most commonly nausea (n = 3, 13.0%). Four (17.3%) experienced grade 3/4 TRAEs: grade 3 peripheral neuropathy, grade 3 asthenia, grade 3 fatigue, and grade 4 lipase increase. No pembrolizumab-related deaths or discontinuations occurred. Conclusion: Pembrolizumab resulted in durable objective response in a subset of patients with heavily pretreated, advanced PD-L1-positive prostate cancer, and its side effect profile was favorable. ClinicalTrials.gov Identifier: NCT02054806.

Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with recurrent carcinoma of the anal canal.

Background: Safety and efficacy of pembrolizumab, a humanized programmed death 1 monoclonal antibody, was assessed in KEYNOTE-028, a multicohort, phase Ib trial for patients with programmed death ligand 1 (PD-L1)-positive advanced solid tumors. We report results for the cohort of patients with advanced anal carcinoma. Patients and methods: Patients with PD-L1-positive tumors (≥1%) received intravenous pembrolizumab 10 mg/kg once every 2 weeks for up to 2 years or until confirmed progression or unacceptable toxicity. Response was assessed every 8 weeks for the first 6 months and every 12 weeks thereafter per Response Evaluation Criteria In Solid Tumors, version 1.1. Primary endpoints were safety and overall response rate per investigator review. Secondary endpoints included progression-free survival, overall survival, and response duration. Data cutoff date was 1 July 2015. Results: Of the 43 patients with advanced anal carcinoma evaluable for PD-L1 expression, 32 (74%) had PD-L1-positive tumors as assessed with the 22C3 prototype assay, of whom 25 were enrolled between April and September 2014. Sixteen patients (64%) experienced treatment-related adverse events; the most common ones were diarrhea and fatigue in four patients (16%) each and nausea in three patients (12%). There were no treatment-related deaths or discontinuations as of the data cutoff date. Among the 24 patients with squamous cell carcinoma histology, four had confirmed partial response, for an overall response rate of 17% [95% confidence interval (CI), 5%-37%) and 10 (42%) had confirmed stable disease, for a disease control rate of 58%. One additional patient with non-squamous histology had confirmed stable disease. Conclusion: In this population of patients with PD-L1-positive advanced squamous cell anal carcinoma, pembrolizumab demonstrated a manageable safety profile and encouraging antitumor activity. These data support further study of pembrolizumab for this patient population. ClinicalTrials.gov: NCT02054806.

Study of η and η' Photoproduction at MAMI.

The reactions γp→ηp and γp→η^{'}p are measured from their thresholds up to the center-of-mass energy W=1.96 GeV with the tagged-photon facilities at the Mainz Microtron, MAMI. Differential cross sections are obtained with unprecedented statistical accuracy, providing fine energy binning and full production-angle coverage. A strong cusp is observed in the total cross section for η photoproduction at the energies in the vicinity of the η^{'} threshold, W=1896 MeV (E_{γ}=1447 MeV). Within the framework of a revised ηMAID isobar model, the cusp, in connection with a steep rise of the η^{'} total cross section from its threshold, can only be explained by a strong coupling of the poorly known N(1895)1/2^{-} state to both ηp and η^{'}p. Including the new high-accuracy results in the ηMAID fit to available η and η^{'} photoproduction data allows the determination of the N(1895)1/2^{-} properties.

Measurement of the transverse target and beam-target asymmetries in η meson photoproduction at MAMI.

We present new data for the transverse target asymmetry T and the very first data for the beam-target asymmetry F in the γ[over →]p[over →]→ηp reaction up to a center-of-mass energy of W=1.9 GeV. The data were obtained with the Crystal-Ball/TAPS detector setup at the Glasgow tagged photon facility of the Mainz Microtron MAMI. All existing model predictions fail to reproduce the new data indicating a significant impact on our understanding of the underlying dynamics of η meson photoproduction. The peculiar nodal structure observed in existing T data close to threshold is not confirmed.

Adjuvant immunotherapy for melanoma patients: progress and opportunities.

The majority of patients who are diagnosed with cutaneous melanoma are candidates for surgical resection and thus curable from their disease. However, the risk for a recurrence is high for many patients, including those with lymph node-negative melanoma, thus necessitating additional therapies beyond surgery. With the advent of anti-programmed cell death protein 1 (PD-1)-based immunotherapies, which are vastly more effective compared to previous standard-of-care treatments in the advanced setting, the landscape of adjuvant therapy has fundamentally changed in recent years. Anti-PD-1-based immune checkpoint inhibition therapy is now the standard of care for many patients with stage IIB or higher melanoma. Neoadjuvant approaches have demonstrated superior outcomes compared to adjuvant-alone therapy. However, a number of questions remain including treatment combinations such as combined anti-PD-1 + lymphocyte activation gene-3, optimal sequencing of therapies, and the use of predictive markers to further improve outcomes for patients with high-risk melanoma.

[Immediate results of combined treatment for soft tissue sarcomas including intraoperative radiation therapy].

Nowadays the most popular and justified, from oncological positions, method of treatment for soft tissue sarcomas is a combined approach with the use of conservative surgery followed by postoperative radiation therapy. In this regard, intraoperative radiation therapy (IORT) in a single dose of 10-20 Gy is a method that optimizes the role of radiation therapy in treatment of this pathology allowing precise localization of radiation zone within the "tumor bed", thereby minimizing damage of normal tissues and critical organs. The aim of the study was to investigate the effect of IORT on the frequency and structure of post-operative complications. Testing group (n = 49) was compared to the group without IORT (n = 57) and group with only surgery (n = 171). According to the study it was not obtained statistically significant differences in the incidence of postoperative complications in the groups (p = 0,57), not marked influence on the structure of post-operative complications.

[Long-term results of combined treatment of soft tissue sarcoma with the use of intraoperative radiotherapy].

This retrospective study of long-term results of combined treatment of soft tissue sarcoma with the use of intraoperative radiotherapy (IORT) included 171 patients. Controls received adjuvant distant gamma-therapy (n = 57) or surgical treatment (n = 171). IORT significantly improved 5 year general (p = 0.025) and relapse-free (p < 0.025) survival rate and the same parameters in patients with a tumour larger than 5 cm (p = 0.0001). The intergroup difference regardless of tumour differentiation was insignificant (p =0.33), but survival rate tended to decrease in patients with moderately and weakly differentiated sarcomas (G2-3). It is concluded that combined treatment with IORT may be used as an alternative method for the treatment of moderately and weakly differentiated sarcomas.

[Results of combined treatment with intraoperative radial therapy of soft tissue sarcoma].

The intraoperative radiation therapy (IORT) in combined therapy of soft tissue sarcoma in a single dose 10-20 Gy is the method, which can optimize the role of radiation therapy in treatment of this nosology. This method allows exact localization of the irradiation zone in the frames of "tumor bed", thereby minimizing the damage of normal tissues and critical organs. The aim of the study was the influence of IORT on the rate and structure of postoperative complications and long-term results of treatment in the group under study (n = 49) in comparison with the group of combined treatment without IORT (n = 57) and the group of surgical treatment. No statistically reliable difference in the rate of postoperative complications in groups (p = 0.57) was obtained and there was no influence on the structure of postoperative complications. At the same time the statistically reliable increase of general survival rates (p = 0.025) and the survival without relapse in the main group (p < 0.025) were obtained. Thus, the application of IORT in combined treatment of soft tissue sarcomas showed the satisfactory profile of "surgical safety", provided the reliable increase of general survival rates and rates without relapse.

Molecular sexing of unusually large numbers of Spheniscus magellanicus (Spheniscidae) washed ashore along the Brazilian coast in 2008.

There have been few studies on Magellanic penguins (Spheniscus magellanicus). In 2008, these penguins washed ashore along the Brazilian coast in unusually high numbers, some reaching as far as northeast Brazil. As Magellanic penguins show little sexual dimorphism, sex determination by morphological features is not accurate. Here, we tested a molecular procedure for sexing specimens of S. magellanicus washed ashore along the coasts of Sergipe, Rio de Janeiro and Rio Grande do Sul in 2008, comparing the sex ratio between these localities. Tissue samples were collected from 135 dead, beached specimens. We carried out total genomic DNA extraction and CHD-Z/CHD-W gene amplification by PCR using P2 and P8 primers. Amplicons were separated by 12% acrylamide gel electrophoresis. We found a greater proportion of females (70%). Sex could be determined because females have two intronic regions of CHD gene of different size in the sex chromosomes, visualized as two bands on the gel (380 and 400 bp approximately), while males have only one (400 bp). Therefore, this method proved to be effective and sensitive for sex determination of S. magellanicus individuals. Data on sex ratios are useful for understanding the dynamics and ecology of Magellanic penguin populations.

Hepatic lactate uptake versus leg lactate output during exercise in humans.

The exponential rise in blood lactate with exercise intensity may be influenced by hepatic lactate uptake. We compared muscle-derived lactate to the hepatic elimination during 2 h prolonged cycling (62 +/- 4% of maximal O(2) uptake, (.)Vo(2max)) followed by incremental exercise in seven healthy men. Hepatic blood flow was assessed by indocyanine green dye elimination and leg blood flow by thermodilution. During prolonged exercise, the hepatic glucose output was lower than the leg glucose uptake (3.8 +/- 0.5 vs. 6.5 +/- 0.6 mmol/min; mean +/- SE) and at an arterial lactate of 2.0 +/- 0.2 mM, the leg lactate output of 3.0 +/- 1.8 mmol/min was about fourfold higher than the hepatic lactate uptake (0.7 +/- 0.3 mmol/min). During incremental exercise, the hepatic glucose output was about one-third of the leg glucose uptake (2.0 +/- 0.4 vs. 6.2 +/- 1.3 mmol/min) and the arterial lactate reached 6.0 +/- 1.1 mM because the leg lactate output of 8.9 +/- 2.7 mmol/min was markedly higher than the lactate taken up by the liver (1.1 +/- 0.6 mmol/min). Compared with prolonged exercise, the hepatic lactate uptake increased during incremental exercise, but the relative hepatic lactate uptake decreased to about one-tenth of the lactate released by the legs. This drop in relative hepatic lactate extraction may contribute to the increase in arterial lactate during intense exercise.

[Limb salvage for the diabetic foot - disease management program]. / Extremitätenerhalt für den diabetischen Fuss - Disease-Management-Programm.

OBJECTIVE: Lower-extremity amputation (LEA) is a common complication among patients with diabetes. This study tests the effects of a structured disease management program for the diabetic foot (DF) aiming to reduce the number of LEA. DESIGN, METHODS: In a prospective study design we investigate patients with DF in a system of outpatient treatment, acute in-patient care and rehabilitative treatment. Subjects were recruited since January 1(st), 2000, with the latest admission being December 31, 2004. All study participants undergo a five-year follow-up observation period. The University of Texas Wound Classification System (UT) of foot ulcers serves as basis of the documentation and analysis. We evaluated numbers of LEA, rates of ulcer healing and underlying forms of peripheral vascular disease. RESULTS: We report the results of the first patient group completing the two-year follow-up examination. In 2000, 102 subjects with new foot ulcers were consecutively included into the study. 68.6% were men, the mean age of the study population was 68.1 +/- 11.4 years and the mean diabetes duration was 19.4 +/- 10.3 years. After two years, 68 patients can still be examined. Altogether, 22 patients (21.6%) died, and 12 (11.8%) dropped out for various reasons. At the point of discharge from the clinics 35.3% of the ulcers had healed and another 44.1% were in UT grade 1. After two years, a complete healing could still be determined with 51 patients (50.0% of the cohort of the original 102 patients, or 75.0% of the subjects reaching the two-year follow-up). 10 subjects (9.8% or 14.5%) were in the UT grade 1. Eight diabetics underwent major amputation (MA) during the two-year examination period (amputation rate 7.8%). CONCLUSIONS: The primary objective of the study, a significant reduction of MA with DF patients, has been achieved. The ulcer healing rates are comparable to the reports of leading centers.

Opposite effect of Cu(II) and Se(IV) ions on the antibacterial-toxic action of mycotoxins.

The effect of Se(IV) and Cu(II) ions on the antibacterial activity of aflatoxins and ochratoxin A (mycotoxins) was studied in BioArena as a complex bioautographic system. In the presence of 0.23 and 0.46 mg/100 mL Se(IV) the inhibition zones of mycotoxins were decreased, however, lower concentration (0.046 mg/100 mL) increased the antibacterial effect of aflatoxin B1. Cu(II) (1.53 mg/100 mL) enhanced the toxicity of mycotoxins. The results supported the possible role of formaldehyde and its reaction products (e.g. 1O2, O3) in the antibacterial-toxic action of mycotoxins. Cu(II) can probably generate and mobilise the formaldehyde molecules and so it could increase the toxicity with its potential reaction products. It is possible that the enzymatic or spontaneous methylation of Se(IV) takes place through formaldehyde, which may cause partial formaldehyde depletion in the system. The enhanced antibacterial effect at low concentration Se(IV) is overlapping with the often experienced prooxidant effect in cases of natural antioxidants.

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane.

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.

The influence of cellular ATP levels on dimyristoylphosphatidylcholine-induced release of vesicles from human erythrocytes.

Release of membrane vesicles from human erythrocytes was induced by modulation of red cell ATP levels, by incubation of erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) suspensions, or by a sequential combination of both procedures. When red blood cell ATP levels were decreased prior to incubation with DMPC, the lag-time between addition of the lipid and beginning of vesiculation was reduced. Furthermore, the rate of vesicle release itself was accelerated. Experiments carried out with a rapid ATP depletion technique showed that the onset of vesiculation and the release were most evidently accelerated in those cases where echinocytes had been formed prior to the addition of DMPC. The results suggest that red blood cells with reduced cellular ATP levels or an altered cell shape are more susceptible to a further perturbation of the membrane by addition of exogenous DMPC.

Modulation of red cell vesiculation by protease inhibitors.

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.

Modulation of erythrocyte vesiculation by amphiphilic drugs.

Release of acetylcholinesterase-containing vesicles from human erythrocyte membranes induced by dimyristoylphosphatidylcholine (DMPC) was inhibited by exposure of red cells to cationic amphiphilic drugs like tetracaine, chlorpromazine and primaquine which all are known to induce stomatocyte formation. On the other hand, the process was facilitated when red cells were exposed to crenators like the anionic drugs indomethacin and phenylbutazone or when DMPC was added to calcium-loaded red cells. The results suggest that agents which are known to modulate red cell shape do also influence the vesiculation behavior of the cells.

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts.

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 micrograms. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.

Reconstitution of human erythrocyte membrane acetylcholinesterase in phospholipid vesicles. Analysis of the molecular forms by cross-linking studies.

Unilamellar lipid vesicles were formed upon removal of Triton X-100 with Amberlite XAD-2 from a mixture of egg phosphatidylcholine and Triton-solubilized pure human erythrocyte membrane acetylcholinesterase. A majority of large (230 nm diameter) vesicles together with a minor population of smaller (30 nm diameter) strictures were observed in freeze-fracture electron micrographs. Reconstitution experiments performed with [phenyl-3H(n)]-Triton X-100 showed that only one detergent molecule per 600 lipid molecules was present in the vesicles. Density gradient centrifugation showed co-sedimentation of acetylcholinesterase with the lipid vesicles. About 60% of the incorporated enzyme was directed towards the vesicle exterior and could be partially degraded by papain. Mainly dimeric acetylcholinesterase was found when the reconstituted or, alternatively, the lipid-free but Triton-solubilized enzyme were cross-linked with glutaraldehyde. Aggregates were observed when the detergent-depleted oligomeric forms of the enzyme were cross-linked. The results thus indicate that mainly the dimeric enzyme form is present in a phospholipid environment.

Phospholipid asymmetry in red blood cells and spectrin-free vesicles during prolonged storage.

Erythrocytes and spectrin-free DMPC-induced vesicles released from the cells were incubated for 3 weeks at 6 degrees C under conditions of metabolic ATP-depletion. Phosphatidylserine (PS) asymmetry was monitored during this period by use of the prothrombinase assay. Prothrombinase activities measured at the beginning of the incubation period indicated that approximately 0.06% of PS was located at the outer layer of the red cell membrane, whereas in DMPC-induced vesicles approximately 1.5% the PS was exposed on the outside. After completion of the incubation period PS exposure on the outside of red cells and vesicles was increased by no more than 5-fold. On the other hand, with vesicles prepared with a significantly increased (4-fold) ATP-content to sustain translocase activity, the incubation process resulted in a surprisingly high (20-fold) increase of PS exposure. With vanadate, an inhibitor of the aminophospholipid translocase, included in the incubation medium, the redistribution of PS was even more pronounced. These observations indicate that PS asymmetry in spectrin-free vesicles can not be directly correlated to either ATP content or translocase activity and suggest that besides the aminophospholipid translocase and the membrane skeleton, other mechanisms must be involved in maintaining phospholipid asymmetry.

A monomeric form of human erythrocyte membrane acetylcholinesterase.

Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained.