Event-free survival by residual cancer burden with pembrolizumab in early-stage TNBC: exploratory analysis from KEYNOTE-522.

BACKGROUND: KEYNOTE-522 demonstrated statistically significant improvements in pathological complete response (pCR) with neoadjuvant pembrolizumab plus chemotherapy and event-free survival (EFS) with neoadjuvant pembrolizumab plus chemotherapy followed by adjuvant pembrolizumab in patients with high-risk, early-stage triple-negative breast cancer (TNBC). Prior studies have shown the prognostic value of the residual cancer burden (RCB) index to quantify the extent of residual disease after neoadjuvant chemotherapy. In this preplanned exploratory analysis, we assessed RCB distribution and EFS within RCB categories by treatment group. PATIENTS AND METHODS: A total of 1174 patients with stage T1c/N1-2 or T2-4/N0-2 TNBC were randomized 2 : 1 to pembrolizumab 200 mg or placebo every 3 weeks given with four cycles of paclitaxel + carboplatin, followed by four cycles of doxorubicin or epirubicin + cyclophosphamide. After surgery, patients received pembrolizumab or placebo for nine cycles or until recurrence or unacceptable toxicity. Primary endpoints are pCR and EFS. RCB is a prespecified exploratory endpoint. The association between EFS and RCB was assessed using a Cox regression model. RESULTS: Pembrolizumab shifted patients into lower RCB categories across the entire spectrum compared with placebo. There were more patients in the pembrolizumab group with RCB-0 (pCR), and fewer patients in the pembrolizumab group with RCB-1, RCB-2, and RCB-3. The corresponding hazard ratios (95% confidence intervals) for EFS were 0.70 (0.38-1.31), 0.92 (0.39-2.20), 0.52 (0.32-0.82), and 1.24 (0.69-2.23). The most common first EFS events were distant recurrences, with fewer in the pembrolizumab group across all RCB categories. Among patients with RCB-0/1, more than half [21/38 (55.3%)] of all events were central nervous system recurrences, with 13/22 (59.1%) in the pembrolizumab group and 8/16 (50.0%) in the placebo group. CONCLUSIONS: Addition of pembrolizumab to chemotherapy resulted in fewer EFS events in the RCB-0, RCB-1, and RCB-2 categories, with the greatest benefit in RCB-2. These findings demonstrate that pembrolizumab not only increased pCR rates, but also improved EFS among most patients who do not have a pCR.

A network approach to developing immuno-oncology combinations in Canada.

Immune checkpoint inhibitors have revolutionized care for many cancer indications, with considerable effort now being focused on increasing the rate, depth, and duration of patient response. One strategy is to combine immune strategies (for example, ctla-4 and PD-1/L1-directed agents) to harness additive or synergistic efficacy while minimizing toxicity. Despite encouraging results with such combinations in multiple tumour types, numerous clinical challenges remain, including a lack of biomarkers that reliably predict outcome, the emergence of therapeutic resistance, and optimal management of immune-related toxicities. Furthermore, the selection of ideal combinations from the myriad of immune, systemic, and locoregional therapies has yet to be determined. A longitudinal network-based approach could offer advantages in addressing those critical questions, including long-term follow-up of patients beyond individual trials. The molecular cancer registry Personalize My Treatment, managed by the Networks of Centres of Excellence nonprofit organization Exactis Innovation, is uniquely positioned to accelerate Canadian immuno-oncology (io) research efforts throughout its national network of cancer sites. To gain deeper insight into how a pan-Canadian network could advance research in io combinations, Exactis invited preeminent clinical and scientific advisors from across Canada to a roundtable event in November 2017. The present white paper captures the expert advice provided: leverage longitudinal patient data collection; facilitate network collaboration and assay harmonization; synergize with existing initiatives, networks, and biobanks; and develop an io combination trial based on Canadian discoveries.

Effects of a green tea extract, Polyphenon E, on systemic biomarkers of growth factor signalling in women with hormone receptor-negative breast cancer.

BACKGROUND: Observational and experimental data support a potential breast cancer chemopreventive effect of green tea. METHODS: We conducted an ancillary study using archived blood/urine from a phase IB randomised, placebo-controlled dose escalation trial of an oral green tea extract, Polyphenon E (Poly E), in breast cancer patients. Using an adaptive trial design, women with stage I-III breast cancer who completed adjuvant treatment were randomised to Poly E 400 mg (n = 16), 600 mg (n = 11) and 800 mg (n = 3) twice daily or matching placebo (n = 10) for 6 months. Blood and urine collection occurred at baseline, and at 2, 4 and 6 months. Biological endpoints included growth factor [serum hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)], lipid (serum cholesterol, triglycerides), oxidative damage and inflammatory biomarkers. RESULTS: From July 2007-August 2009, 40 women were enrolled and 34 (26 Poly E, eight placebo) were evaluable for biomarker endpoints. At 2 months, the Poly E group (all dose levels combined) compared to placebo had a significant decrease in mean serum HGF levels (-12.7% versus +6.3%, P = 0.04). This trend persisted at 4 and 6 months but was no longer statistically significant. For the Poly E group, serum VEGF decreased by 11.5% at 2 months (P = 0.02) and 13.9% at 4 months (P = 0.05) but did not differ compared to placebo. At 2 months, there was a trend toward a decrease in serum cholesterol with Poly E (P = 0.08). No significant differences were observed for other biomarkers. CONCLUSIONS: Our findings suggest potential mechanistic actions of tea polyphenols in growth factor signalling, angiogenesis and lipid metabolism.

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis.

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.

Production of 6-deoxy-13-cyclopropyl-erythromycin B by Saccharopolyspora erythraea NRRL 18643.

Cyclopropane carboxylic acid was fed to Saccharopolyspora erythraea NRRL 18643 (6-deoxyerythromycin producer), resulting in the production of 6-deoxy-13-cyclopropyl-erythromycin B. These studies provide further evidence that deoxyerythronolide B synthase has a relaxed specificity for the starter unit.

Novel erythromycins from a recombinant Saccharopolyspora erythraea strain NRRL 2338 pIG1. I. Fermentation, isolation and biological activity.

In a previous report, a plasmid, pIG1, which contained the loading domain from the Streptomyces avermitilis polyketide synthase (PKS), promoters from Streptomyces coelicolor and the DEBS1-TE truncated PKS from Saccharopolyspora erythraea, was integrated into the S. erythraea chromosome, effectively replacing the natural erythromycin loading domain with the avermectin loading domain. In this paper, we report the feeding of short-chained fatty acids to this recombinant strain, and its parent, NRRL 2338. Both strains incorporated exogenously supplied fatty acids to produce novel, biologically active, C-13 substituted erythromycins.

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes.

The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyrl-CoA and isobutyrl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyrl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA.

A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins.

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described. The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.

Composition and Rheological Properties of Extracellular Polysaccharide 105-4 Produced by Pseudomonas sp. Strain ATCC 53923.

A soil isolate produced a novel extracellular polysaccharide (EPS) with unusually potent thickening powers. The EPS contained d-mannose, d-glucose, d-galactose, and d-glucuronic acid in the unique molar ratio 1:4:1:2 and 10 to 15% acetate. Viscosities of a 1-g/liter aqueous solution were 1 x 10 and 14 x 10 cP at shear rates of 0.01 and 0.1 s, respectively. The EPS was insensitive to high concentrations of NaCl and CaCl(2).

Skin mechanics measured in vivo using torsion: a new and accurate model more sensitive to age, sex and moisturizing treatment.

Summary Measurements of skin mechanics are required to understand better cracking and flaking of the epidermis and loss of 'elasticity'with age in the dermis. Improvements in torsional testing are described here. The resulting data was fitted to algebraic models, the parameters of which can serve both as a concise description of the responses and as a means of relating them to skin structure and physiology. This investigation looks into the suitability of seven such algebraic models. Five of the models examined here appear to be new. Using the commercially available Dia-Stron DTM Torque Meter with our own software, model parameters were studied as indicators of the effects of age and sex in 41 people, and of skin moisturizing treatments in a further 10 people. The two models in the literature were both found to be substantially less accurate and sensitive representations of experimental data than one of the new models proposed here based on the Weibull distribution. This 'WB model'was consistently the one best able to distinguish differences and detect changes which were statistically significant. The WB model appears to be the most powerful and efficient available. Use of this model makes it possible to demonstrate in vivo a statistically significant mechanical difference between male and pre-menopausal female skin using only one parameter (p= 0.0163, with 18 males and 19 females) and to demonstrate a statistically significant mechanical difference between successive decades of age in female skin using only one parameter (p= 0.0124, n= 24). The two parameters of the model most sensitive to skin structure, function and treatment have been combined to form the axes of a 'Skin condition chart'. Any person can be located on this chart at a point indicating their overall skin condition in mechanical terms and any changes in that condition can be clearly demonstrated by movement across the plot.

Branched-chain fatty acid requirement for avermectin production by a mutant of Streptomyces avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity.

The eight natural avermectins produced by Streptomyces avermitilis have the carbon skeleton of either isobutyric or S-2-methylbutyric acid incorporated into their structures. A mutant of S. avermitilis has been isolated that contains no functional branched-chain 2-oxo acid dehydrogenase activity. The mutant, in contrast to its parent, is unable to grow with isoleucine, valine and leucine as carbon sources. In medium lacking both S(+)-2-methylbutyric and isobutyric acid, the mutant is also incapable of making the natural avermectins, while supplementation with either one of these compounds restores production of the corresponding four natural avermectins. These facts indicate that in S. avermitilis the branched-chain 2-oxo acid dehydrogenase enzyme functions not only to catabolize the cellular branched-chain amino acids in order to meet energy and growth requirements but also to provide the small branched-chain organic acid precursor molecules necessary for avermectin biosynthesis. Supplementation of the mutant strain with R(-)-2-methylbutyric acid yields novel isomeric avermectins unseen in the (unsupplemented) wild-type strain. It was also concluded that acetate and propionate production by branched-chain 2-oxo acid degradation is not absolutely essential for avermectin production.

Association of alginate from Pseudomonas aeruginosa with two forms of heparin-binding lectin isolated from rat lung.

An endogenous heparin-binding lectin activity isolated from rat lung was separated into two distinct isolectin forms which showed subtle changes in carbohydrate specificity. The two lectin forms displayed different specificities toward alginic acid-purified cystic fibrosis isolates of Pseudomonas aeruginosa when assayed by inhibition of both hemagglutination and [3H]heparin binding. This ability of isolectin forms to show higher affinity toward alginic acid from certain P. aeruginosa strains may suggest that there is a selective mechanism in the colonization of patients with cystic fibrosis.

Interaction of a rat lung lectin with the exopolysaccharides of Pseudomonas aeruginosa.

The specific interaction between the exopolysaccharide purified from a number of Pseudomonas aeruginosa isolates from cystic fibrosis patients and a rat lung heparin-lectin was assayed. The polysaccharide prepared from Homma serotypes M, B, I, and G did not act as hapten inhibitors of lectin activity, whereas the polymers prepared from ca. 80% of strains that did not type with Homma serum did act as hapten inhibitors. Inhibition was shown not to be due to lipopolysaccharide. The infrared spectrums of both inhibitory and noninhibitory polymers appeared very similar, although small amounts of glucose and an unidentified amino sugar were found only in the nontypable strains. This evidence suggests that rat lung lectin recognizes and distinguishes a specific type of alginate-like polymer prevalent on the Homma nontypable P. aeruginosa.

Attachment of the main chain to the linkage unit in biosynthesis of teichoic acids.

The main chain of teichoic acids can be assembled in cell-free membrane preparations by the transfer of residues from the appropriate nucleotide precursors to an incompletely characterized amphiphilic molecule, lipoteichoic acid carrier (LTC). However, in the cell wall, the main chain is attached to peptidoglycan through a linkage unit which is synthesized independently. It is believed that, in these cell-free systems, lipid intermediates carrying linkage units are also able to accept residues directly from nucleotide precursors to build up the main chain. In this paper, we have shown that the main chain attached to LTC was transferred from LTC to lipids containing the linkage unit. Thus, in these systems, there appear to be two routes to the biosynthesis of teichoic acid-linkage unit complexes, one by direct assembly of the main chain on linkage unit lipids and the other by transfer of the preassembled main chain from LTC to the linkage unit. It was also shown that linkage unit lipids from different organisms were interchangeable and that these were used for polymer synthesis by Bacillus subtilis 3610, in which the teichoic acid is a poly(glycerol phosphate).