A phase II randomized trial of cobimetinib plus chemotherapy, with or without atezolizumab, as first-line treatment for patients with locally advanced or metastatic triple-negative breast cancer (COLET): primary analysis.

BACKGROUND: Resistance to standard chemotherapy in metastatic triple-negative breast cancer (mTNBC) is associated with upregulation of the mitogen-activated protein kinase (MAPK) pathway. Cobimetinib, an MAPK/extracellular signal-regulated kinase (MEK) inhibitor, may increase sensitivity to taxanes and programmed death-ligand 1 inhibitors. COLET is a three-cohort phase II study evaluating first-line cobimetinib plus chemotherapy, with or without atezolizumab, in patients with locally advanced or mTNBC. PATIENTS AND METHODS: Patients were ≥18 years with locally advanced or mTNBC. Following a safety run-in, patients in cohort I were randomized 1:1 to cobimetinib (60 mg, D3-D23 of each 28-day cycle) or placebo, plus paclitaxel (80 mg/m2, D1, 8, and 15). Additional patients were randomized (1:1) to cohort II or III to receive cobimetinib plus atezolizumab (840 mg, D1 and D15) and either paclitaxel (cohort II) or nab-paclitaxel [cohort III (100 mg/m2, D1, D8, and D15)]. Primary endpoints were investigator-assessed progression-free survival (PFS) (cohort I) and confirmed objective response rate (ORR) (cohorts II/III). Safety and tolerability were also assessed. RESULTS: In the expansion stages, median PFS was 5.5 months for cobimetinib/paclitaxel versus 3.8 months for placebo/paclitaxel in cohort I [hazard ratio 0.73; 95% confidence interval (CI) 0.43-1.24; P = 0.25]. In cohort I, ORR was 38.3% (95% CI 24.40-52.20) for cobimetinib/paclitaxel and 20.9% (95% CI 8.77-33.09) for placebo/paclitaxel; ORRs in cohorts II and III were 34.4% (95% CI 18.57-53.19) and 29.0% (95% CI 14.22-48.04), respectively. Diarrhea was the most common grade ≥3 adverse events across all cohorts. CONCLUSIONS: Cobimetinib added to paclitaxel did not lead to a statistically significant increase in PFS or ORR, although a nonsignificant trend toward a numerical increase was observed. Cobimetinib plus atezolizumab and a taxane did not appear to increase ORR. This demonstrates the potential activity of a combinatorial MEK inhibitor, chemotherapy, and immunotherapy in this difficult-to-treat population.

Port wine stain treatment outcomes have not improved over the past three decades.

BACKGROUND: Since the early '80s, the pulsed dye laser has been the standard treatment tool for non-invasive port wine stain (PWS) removal. In the last three decades, a considerable amount of research has been conducted to improve clinical outcomes, given that a fraction of PWS patients proved recalcitrant to laser treatment. Whether this research actually led to increased therapeutic efficacy has not been systematically investigated. OBJECTIVE: To analyse therapeutic efficacy in PWS patients globally from 1986 to date. METHODS: PubMed was searched for all available PWS trials. Studies with a quartile percentage improvement scale were included, analysed and plotted chronologically. Treatment and patient characteristics were extracted. A mean clearance per study was calculated and plotted. A 5-study simple moving average was co-plotted to portray the trend in mean clearance over time. The data were separately analysed for multiple treatment sessions in previously untreated patients. RESULTS: Sixty-five studies were included (24.3% of eligible studies) comprising 6207 PWS patients. Of all patients, 21% achieved 75-100% clearance. Although a few studies reported remarkably good outcomes in a subset of carefully selected patients, there was no upward trend over time in mean clearance. CONCLUSION: The efficacy of PWS therapy has not improved in the past decades, despite numerous technical innovations and pharmacological interventions. With an unwavering patient demand for better outcomes, the need for development and implementation of novel therapeutic strategies to clear all PWS is as valid today as it was 30 years ago.

Down-regulation of nitric oxide production by droloxifene and toremifene in human breast cancer cells.

We investigated the effect of tamoxifen, 4-OH tamoxifen, toremifene droloxifene, interferon-alpha2a, interferon-alpha2b and interferon-alpha2c, singly and in combination, for their effect on nitric oxide production by MCF-7 and ZR-75-1 human breast cancer cells. Tamoxifen and 4-OH tamoxifen singly had no effect on nitric oxide production by either cell line. However, treatment with droloxifene or toremifene significantly reduced nitric oxide production by both MCF-7 and ZR-75-1 human breast cancer cell lines. Combination treatment with anti-estrogens and interferon-alpha2a interferon-alpha2b or interferon-alpha2c had no synergistic or additive effect compared to each drug singly.

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery.

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth.

Red cell transfusion practice following the transfusion requirements in critical care (TRICC) study: prospective observational cohort study in a large UK intensive care unit.

BACKGROUND AND OBJECTIVES: The Transfusion Requirements In Critical Care (TRICC) study found that critically ill patients tolerate a restrictive haemoglobin transfusion threshold. We investigated red-cell transfusion practice since publication of the TRICC study in a large Scottish teaching hospital intensive care unit (ICU). MATERIALS AND METHODS: We prospectively collected daily data for a 6-month period on haemoglobin concentrations, red-cell transfusions and indications for transfusions, throughout ICU stay for all patients who stayed for longer than 24 h in the ICU. RESULTS: A total of 176 patients were studied, who utilized 1237 ICU days. Of these 176 patients, 52% received red-cell transfusions. A haemoglobin concentration of < or = 9 g/dl was measured in 55% of patients; this occurred by day 1 and day 2 in 52% and 77% of these cases, respectively. Overall the haemoglobin concentration was < or = 9 g/dl for 45% of all patient days. Total red-cell use was 3.1 units per admission (0.47 units per patient day). Only 18% of transfusion episodes were required as a result of haemorrhage. For 'non-haemorrhage' transfusion episodes, the median pretransfusion haemoglobin concentration was 7.8 g/dl (interquartile range: 7.4-8.4 g/dl), and 64% of transfusion episodes were for 2 units. CONCLUSIONS: Clinicians in our centre were conservative, in keeping with recent transfusion guidelines, but deviated from the TRICC protocol by transfusing at haemoglobin concentrations of between 7 and 9 g/dl, rather than below 7 g/dl, and by prescribing 2 unit transfusions. Significant numbers of red-cell units are still used in the critically ill.

The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains.

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.