Early ctDNA dynamics as a surrogate for progression-free survival in advanced breast cancer in the BEECH trial.

BACKGROUND: Dynamic changes in circulating tumour DNA (ctDNA) levels may predict long-term outcome. We utilised samples from a phase I/II randomised trial (BEECH) to assess ctDNA dynamics as a surrogate for progression-free survival (PFS) and early predictor of drug efficacy. PATIENTS AND METHODS: Patients with estrogen receptor-positive advanced metastatic breast cancer (ER+ mBC) in the BEECH study, paclitaxel plus placebo versus paclitaxel plus AKT inhibitor capivasertib, had plasma samples collected for ctDNA analysis at baseline and at multiple time points in the development cohort (safety run-in, part A) and validation cohort (randomised, part B). Baseline sample ctDNA sequencing identified mutations for longitudinal analysis and mutation-specific digital droplet PCR (ddPCR) assays were utilised to assess change in ctDNA abundance (allele fraction) between baseline and 872 on-treatment samples. Primary objective was to assess whether early suppression of ctDNA, based on pre-defined criteria from the development cohort, independently predicted outcome in the validation cohort. RESULTS: In the development cohort, suppression of ctDNA was apparent after 8 days of treatment (P = 0.014), with cycle 2 day 1 (4 weeks) identified as the optimal time point to predict PFS from early ctDNA dynamics. In the validation cohort, median PFS was 11.1 months in patients with suppressed ctDNA at 4 weeks and 6.4 months in patients with high ctDNA (hazard ratio = 0.20, 95% confidence interval 0.083-0.50, P < 0.0001). There was no difference in the level of ctDNA suppression between patients randomised to capivasertib or placebo overall (P = 0.904) nor in the PIK3CA mutant subpopulation (P = 0.071). Clonal haematopoiesis of indeterminate potential (CHIP) was evident in 30% (18/59) baseline samples, although CHIP had no effect on tolerance of chemotherapy nor on PFS. CONCLUSION: Early on-treatment ctDNA dynamics are a surrogate for PFS. Dynamic ctDNA assessment has the potential to substantially enhance early drug development. CLINICAL REGISTRATION NUMBER: NCT01625286.

A colourimetric evaluation of the effect of bacterial contamination on teeth stained with blood in vitro: Evaluation of the efficacy of two different bleaching regimes.

BACKGROUND: Tooth discolouration could occur due to bacterial contamination in traumatized teeth. Hydrogen peroxide is the commonly used bleaching agent. However, due to concerns over safety, alternative bleaching regimes such as sodium perborate (S) and thiourea-hydrogen peroxide (T) have been investigated. METHODS: Apices resected and pulp extirpated 99 premolars were divided into two groups. Group 1 and 2 was injected with blood and blood/bacteria, stored anaerobically for 35 days. The two groups were treated by bleaching with water, S or T. Teeth were rebleached after 7 days. Colourimetric evaluation was assessed using digital imaging, CasMatch standardization and CIE L*a*b colour system preoperatively, 35 days of staining and 7 and 14 of bleaching. A linear mixed model with fixed effects of time, group and bleach was used to examine colour difference. RESULTS: Blood-stained teeth were significantly redder and darker on day 35 compared with blood/bacteria-stained teeth. After bleaching, blood-stained teeth retained significant redness compared with blood/bacteria-stained teeth using either S or T. T produced a significantly whiter shade in both the groups after 14 days. CONCLUSIONS: Blood-stained teeth were significantly darker and red compared with blood/bacteria-stained teeth. T bleaching regime was more effective than S.

Investigation of the effect of rapid and slow external pH increases on Enterococcus faecalis biofilm grown on dentine.

BACKGROUND: Calcium hydroxide is a common endodontic medicament and has an antimicrobial effect by increasing the localized pH within the root canal. However, Enterococcus faecalis has shown some resistance to calcium hydroxide. METHODS: A flow cell apparatus was used to grow an E. faecalis biofilm on dentine discs. Following 4 weeks growth in Todd Hewitt Broth, flow cells were exposed to either a rapid or slow increase to pH 11.5 or 12.5. Cellular viability was determined using serial plating and the number of colony-forming units was normalized against the cellular protein content. Scanning electron microscopy was carried out to qualitatively observe the effects of the different rates of pH increase. RESULTS: A significant difference in viability between the pH rapid and slow groups was not shown in this study. Compared with pH 11.5 solutions, pH 12.5 solutions were more effective at killing bacteria although some E. faecalis still survived. CONCLUSIONS: Enterococcus faecalis did not adapt and develop a greater resistance to high pH following a slow rise in pH compared with a rapid rise in pH. As expected, pH 12.5 was more effective in reducing bacterial numbers compared with pH 11.5 although E. faecalis was not completely eliminated.

Comparative efficacy of endodontic medicaments and sodium hypochlorite against Enterococcus faecalis biofilms.

BACKGROUND: Many studies have investigated the effectiveness of root canal irrigants and medicaments against Enterococcus faecalis. The aim of this study was to compare the efficacy of commonly used medicaments against E. faecalis cultured as a biofilm on dentine substrate. METHODS: An E. faecalis biofilm was established on human dentine slices using a continuous flow cell. Each test medicament (Ledermix, Ca(OH)2 , Odontopaste, 0.2% chlorhexidine and 50:50 combinations of Ledermix/Ca(OH)2 and Odontopaste/Ca(OH)2 ) was introduced into the flow cell and biofilms were harvested and quantitated by determining cellular protein. Cellular viability was determined using serial plating and the number of colony-forming units was normalized against cellular protein to allow treatment protocols to be compared. Qualitative scanning electron microscopy analyses of the biofilm were performed after a 48-h exposure to each test agent. RESULTS: Sodium hypochlorite achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm. Ca(OH)2 and 50:50 combinations of Ca(OH)2 /Ledermix or Ca(OH)2 /Odontopaste reduced the viability by more than 99% while 0.2% chlorhexidine reduced bacterial numbers by 97%. CONCLUSIONS: Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. However, Ca(OH)2 in isolation and combined with Ledermix, and Odontopaste was highly effective in reducing bacterial viability.

A randomized, open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification.

BACKGROUND: Approximately 5%-10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization. PATIENTS AND METHODS: Patients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m2 administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken. RESULTS: Of 71 patients randomized (AZD4547 n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547 n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression. CONCLUSIONS: AZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.

Pertuzumab plus trastuzumab in combination with standard neoadjuvant anthracycline-containing and anthracycline-free chemotherapy regimens in patients with HER2-positive early breast cancer: a randomized phase II cardiac safety study (TRYPHAENA).

BACKGROUND: Pertuzumab (P) combined with trastuzumab (H)-based chemotherapy improves efficacy in early and advanced HER2-positive breast cancer. We assessed the tolerability, with particular focus on cardiac safety, of H and P with chemotherapy in the neoadjuvant treatment of HER2-positive early breast cancer. PATIENTS AND METHODS: In this multicenter, open-label phase II study, patients with operable, locally advanced, or inflammatory breast cancer were randomized 1 : 1 : 1 to receive six neoadjuvant cycles q3w (Arm A: 5-fluorouracil, epirubicin, cyclophosphamide [FEC] + H + P ×3 → docetaxel [T] + H + P ×3; Arm B: FEC ×3 → T + H + P ×3; Arm C: T + carboplatin + H [TCH]+P ×6). pCR was assessed at surgery and adjuvant therapy given to complete 1 year of H. RESULTS: Two hundred twenty-five patients were randomized. During neoadjuvant treatment, two patients (2.7%; Arm B) experienced symptomatic left ventricular systolic dysfunction (LVSD) and 11 patients (Arm A: 4 [5.6%]; Arm B: 4 [5.3%]; Arm C: 3 [3.9%]) had declines in left ventricular ejection fraction of ≥10% points from baseline to <50%. Diarrhea was the most common adverse event. pCR (ypT0/is) was reported for 61.6% (Arm A), 57.3% (Arm B), and 66.2% (Arm C) of patients. CONCLUSION: The combination of P with H and standard chemotherapy resulted in low rates of symptomatic LVSD.

Characterization of three intermediates in the biosynthesis of teichuronic acid of Micrococcus luteus.

Teichuronic acid, the Micrococcus luteus cell wall polysaccharide which consists of D-glucose and N-acetyl-D-mannosaminuronic acid, is synthesized in vitro from uridine diphosphate N-acetyl-D-glucosamine, uridine diphosphate N-acetyl-D-mannosaminuronic acid, and uridine diphosphate D-glucose in a series of reactions catalyzed by a particulate enzyme preparation. Several lipid-linked intermediates are formed, of which the first three are called components A, B, and C. The formation of these intermediates is inhibited by tunicamycin. The lipid moiety of the intermediates is approximately 95% undecaprenol and 5% dodecaprenol as determined by mass spectrometry. The oligosaccharide moieties of components B and C are the disaccharide, N-acetyl-D-mannosaminuronyl-(1,3)-N-acetyl-D-glucosamine, and the trisaccharide, N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminuronyl++ +-(1, 3)-N-acetyl-D-glucosamine, respectively, as determined by the complete degradation of the former and partial degradation of the latter by the alkaline beta-elimination reaction. The saccharide and lipid moieties of the intermediates are linked through pyrophosphate. Thus, component A is P1-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, component B is P1-N-acetyl-D-mannosaminuronyl-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, and component C is P1-N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminurony l-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate.

Glutathione-dependent reductive dehalogenation of 2,2',4'-trichloroacetophenone to 2',4'-dichloroacetophenone.

alpha-Haloketones are highly reactive compounds, which are known to undergo enzymatic reduction to methyl ketones. The objective of this research was to characterize the enzymes involved in this reaction and to investigate the mechanism of the reaction. 2,2',4'-Trichloroacetophenone was reduced to 2',4'-dichloroacetophenone by glutathione-dependent cytosolic enzymes present in the liver, kidney, and brain. The actual substrate for the enzyme was S-(2,4-dichlorophenacyl)glutathione, which is formed by the nonenzymic reaction of 2,2',4'-trichloroacetophenone and glutathione. The reaction mechanism may involve an enzyme-catalyzed nucleophilic attack of glutathione on the sulfur atom of S-(2,4-dichlorophenacyl)glutathione to yield a carbanion and glutathione disulfide; protonation of the carbanion would yield 2',4'-dichloroacetophenone. Stoichiometry studies showed that the glutathione disulfide/2',4'-dichloroacetophenone ratio was 1.25 +/- 0.13.

Thioredoxin-dependent sulfoxide reduction by rat renal cytosol.

The reduction of sulindac to sulindac sulfide by rat renal enzymes has been characterized. This biotransformation is catalyzed by cytosolic enzymes requiring NADPH. The KM and Vmax for sulindac were 45.0 +/- 13.1 microM and 0.48 +/- 0.15 nmol of sulindac sulfide per 5 mg of protein per min, respectively. The reaction was inhibited by sulfhydryl reagents and several sulfoxides. Furthermore, disulfides [insulin, glutathione disulfide, L-cystine, and 5,5'-dithiobis (2-nitrobenzoic acid)] known to interact with thioredoxin-dependent enzyme systems inhibited sulindac reduction, as did sodium arsenite, a known inhibitor of thioredoxin reductase. Removal of thioredoxin from renal cytosolic fractions by gel-filtration chromatography resulted in a marked diminution of sulindac reduction: activity was restored by addition of purified Escherichia coli thioredoxin or dithiothreitol. These findings demonstrate the involvement of thioredoxin in renal sulfoxide reduction.

Sulfoxide reduction. In vitro reduction of sulindac by rat hepatic cytosolic enzymes.

The reductive biotransformation of sulindac to the corresponding sulfide has been characterized in vitro in rat preparations with respect to subcellular and tissue distribution, cofactor requirements, kinetic constants, and the effects of inhibitors. The highest activity was found in hepatic and renal cytosolic fractions. Either NADPH or dithiothreitol effectively supported the reaction but NADH was much less effective. The KM and Vmax for sulindac were found to be 36.7 +/- 3.8 microM and 75.7 +/- 9.7 pmol/min/mg of protein, respectively, and the KM and Vmax for NADPH were found to be 0.48 +/- 0.03 microM and 43.3 +/- 4.6 pmol/min/mg of protein, respectively. The most effective inhibitors of the reaction were sulfhydryl reagents and sulfoxides structurally related to sulindac.