Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Association of post-operative ctDNA detection with outcomes of patients with early breast cancers.

BACKGROUND: In early breast cancer (EBC) patients, we aimed to determine whether circulating tumor DNA (ctDNA) analysis following primary surgery, before systemic therapy, identified molecular residual disease and was associated with risk of relapse and relapse-free survival (RFS). METHODS: Plasma was collected, retrospectively, before surgery, 1-14 weeks post-operatively, and before adjuvant therapy, and in a subset of patients after adjuvant therapy. A personalized, tumor-informed, multiplex PCR next generation sequencing assay (Signatera™) was used for ctDNA detection and quantification. The primary objective was to compare RFS and distant recurrence-free survival (DRFS) in patients with detected versus non-detected ctDNA. RESULTS: A total of 48 patients with EBC (median age 50.5 years) [34 hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-), 5 HER2+, 9 triple-negative breast cancer) were included. ctDNA was detected in 64.5% (20/31) of patients before surgery, and 35.4% (17/48) after surgery. ctDNA detection before surgery was associated with tumor grade (P = 0.019), ctDNA detection after surgery was associated with receptor subtype (P = 0.01). Patients with ctDNA detected after surgery had worse DRFS [hazard ratio = 5.5, 95% confidence interval (CI) 1.1-28.5, P = 0.04]. RFS in patients with ctDNA detected after surgery was worse than in those with lack of ctDNA detection, although not statistically significant (hazard ratio = 3.7, 95% CI 0.9-15.7, P = 0.073). Patients with ctDNA detected preoperatively or post-operatively had a trend towards worse RFS (hazard ratio = 7.8, 95% CI 0.9-63.7, P = 0.05) and DRFS (hazard ratio = 6.8, 95% CI 0.8-57, P = 0.07) compared with those with ctDNA undetected at both timepoints. ctDNA detection anticipated clinical relapse with a median lead time of 16 months. CONCLUSIONS: In patients with treatment-naive EBC, ctDNA is detectable after surgery. The absence of ctDNA at a single post-surgical timepoint is associated with improved DRFS, supporting the development of future trials studying de-escalation of systemic therapy.

The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate.

BACKGROUND: Hexokinase I is the pacemaker of glycolysis in brain tissue. The type I isozyme exhibits unique regulatory properties in that physiological levels of phosphate relieve potent inhibition by the product, glucose-6-phosphate (Gluc-6-P). The 100 kDa polypeptide chain of hexokinase I consists of a C-terminal (catalytic) domain and an N-terminal (regulatory) domain. Structures of ligated hexokinase I should provide a basis for understanding mechanisms of catalysis and regulation at an atomic level. RESULTS: The complex of human hexokinase I with glucose and Gluc-6-P (determined to 2.8 A resolution) is a dimer with twofold molecular symmetry. The N- and C-terminal domains of one monomer interact with the C- and N-terminal domains, respectively, of the symmetry-related monomer. The two domains of a monomer are connected by a single alpha helix and each have the fold of yeast hexokinase. Salt links between a possible cation-binding loop of the N-terminal domain and a loop of the C-terminal domain may be important to regulation. Each domain binds single glucose and Gluc-6-P molecules in proximity to each other. The 6-phosphoryl group of bound Gluc-6-P at the C-terminal domain occupies the putative binding site for ATP, whereas the 6-phosphoryl group at the N-terminal domain may overlap the binding site for phosphate. CONCLUSIONS: The binding synergism of glucose and Gluc-6-P probably arises out of the mutual stabilization of a common (glucose-bound) conformation of hexokinase I. Conformational changes in the N-terminal domain in response to glucose, phosphate, and/or Gluc-6-P may influence the binding of ATP to the C-terminal domain.

Refined crystal structures of glucoamylase from Aspergillus awamori var. X100.

The refined crystal structures of a proteolytic fragment of glucoamylase from Aspergillus awamori var. X100 have been determined at pH 6 and 4 to a resolution of 2.2 A and 2.4 A, respectively. The models include the equivalent of residues 1 to 471 of glucoamylase from Aspergillus niger and a complete interpretation of the solvent structure. The R-factors of the pH 6 and 4 structures are 0.14 and 0.12, respectively, with root-mean-square deviations of 0.014 A and 0.012 A from expected bondlengths. The enzyme has the general shape of a doughnut. The "hole" of the doughnut consists of a barrier of hydrophobic residues at the center, which separates two water-filled voids, one of which serves as the active site. Three clusters of water molecules extend laterally from the active site. One of the lateral clusters connects the deepest recess of the active site to the surface of the enzyme. The most significant difference in the pH 4 and 6 structures is the thermal parameter of water 500, the putative nucleophile in the hydrolysis of maltooligosaccharides. Water 500 is associated more tightly with the enzyme at pH 4 (the pH of optimum catalysis) than at pH 6. In contrast to water 500, Glu179, the putative catalytic acid of glucoamylase, retains the same conformation in both structures and is in an environment that would favor the ionized, rather than the acid form of the side-chain. Glycosyl chains of 5 and 8 sugar residues are linked to Asparagines 171 and 395, respectively. The conformations of the two glycosyl chains are similar, being superimposable on each other with a root-mean-square discrepancy of 1.9 A. The N-glycosyl chains hydrogen bond to the surface of the protein through their terminal sugars, but otherwise do not interact strongly with the enzyme. The structures have ten serine/threonine residues, to each of which is linked a single mannose sugar. The structure of the ten O-glycosylated residues taken together suggests a well-defined conformation for proteins that have extensive O-glycosylation of their polypeptide chain.

Crystallization and preliminary X-ray study of minor glucoamylase from Aspergillus awamori variant X-100/D27.

Crystals of the reduced form of glucoamylase were obtained from polyethylene glycol 6000 solution by the hanging-drop method. The protein was treated with alpha-mannosidase to partly remove the sugar component. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 116.7 A, b = 104.3 A, c = 48.5 A and diffract beyond 2.5 A resolution.

Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate.

Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 A resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed.

Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation.

Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of two structurally similar halves connected by an alpha-helix. The enzyme dimerizes at elevated protein concentrations in solution and in crystal structures; however, almost all published data reflect the properties of a hexokinase I monomer in solution. Crystal structures of mutant forms of recombinant human hexokinase I, presented here, reveal the enzyme monomer for the first time. The mutant hexokinases bind both glucose 6-phosphate and glucose with high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain. Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosphate reveals a second binding site for adenine nucleotides at the putative active site (C-half), with conformational changes extending 15 A to the contact interface between the N and C-halves. The structures reveal distinct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis.

Crystallization and preliminary X-ray analysis of human brain hexokinase.

Human brain hexokinase type I, expressed in Escherichia coli, has been crystallized from polyethylene glycol 8000 in the presence of inorganic phosphate. The crystals are hexagonal needles of diameter 0.25 mm, diffracting to a resolution of 3.5 A on a rotating-anode/area-detector system. The crystals belong to the space group P3(1)21/P3(2)21 with cell dimensions a = b = 171.5 A and c = 99.4 A. The specific volume of the crystal is 4.2 A3/Da, suggesting an asymmetric unit with a single 100 kDa molecule and a solvent content of 71% by volume or two molecules of hexokinase with a solvent content of 41%. The complex of hexokinase with glucose crystallizes under similar conditions, giving crystals of the same morphology.

Multiple crystal forms of hexokinase I: new insights regarding conformational dynamics, subunit interactions, and membrane association.

Hexokinase I is comprised of homologous N- and C-terminal domains, and binds to the outer membrane of mitochondria. Reported here is the structure of a new crystal form of recombinant human hexokinase I, which complements existing crystal structures. Evidently, in some packing environments and even in the presence of glucose and glucose 6-phosphate the N-terminal domain (but not the C-terminal domain) can undergo oscillations between closed and partially opened conformations. Subunit interfaces, present in all known crystal forms of hexokinase I, promote the formation of linear chains of hexokinase I dimers. Presented is a model for membrane-associated hexokinase I, in which linear chains of hexokinase I dimers are stabilized by interactions with mitochondrial porin.

Refined structure for the complex of D-gluco-dihydroacarbose with glucoamylase from Aspergillus awamori var. X100 to 2.2 A resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase.

The crystal structure at pH 4 of the complex of glucoamylase II(471) from Aspergillus awamori var. X100 with the pseudotetrasaccharide D-gluco-dihydroacarbose has been refined to an R-factor of 0.125 against data to 2.2 A resolution. The first two residues of the inhibitor bind at a position nearly identical to those of the closely related inhibitor acarbose in its complex with glucoamylase at pH 6. However, the electron density bifurcates beyond the second residue of the D-gluco-dihydroacarbose molecule, placing the third and fourth residues together at two positions in the active site. The position of relatively low density (estimated occupancy of 35%) corresponds to the location of the third and fourth residues of acarbose in its complex with glucoamylase at pH 6. The position of high density (65% occupancy) corresponds to a new binding mode of an extended inhibitor to the active site of glucoamylase. Presented are possible causes for the binding of D-gluco-dihydroacarbose in two conformations at the active site of glucoamylase at pH 4.

[NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. II. Enzyme conformation at 3.0 A resolution]. / NAD-zavisimaia formiatdegidrogenaza metilotrofnykh bakterii Pseudomonas sp. 101. II. Prostranstvennaia struktura fermenta pri razreshenii 3,0 A.

Three heavy atom isomorphous derivatives were used for the X-ray analysis of the holo form of NAD-dependent bacterial formate dehydrogenase (ternary complex enzyme-NAD-azide) at 3.0 A resolution. The enzyme subunit contains a catalytic and a coenzyme binding domain, with the active centre and the coenzyme binding site in the cleft between the domains. The polypeptide chain's fold and the position of 393 C alpha-atoms were determined. The secondary structure of the formate dehydrogenase was resolved. The structure of the NAD-binding domain is shown to be similar to that of other NAD-dependent enzymes.

[NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. III. Comparative analysis]. / NAD-zavisimaia formiatdegidrogenaza metilotrofnykh bakterii Pseudomonas sp. 101. III. Sravnitel'nyi analiz.

The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.

[The dispersion analysis of cell suspensions by a conductometric method]. / Analiz dispersnosti kletochnykh suspenzii konduktometricheskim metodom.

A conductometric device-analyser ADS-05 designed by the authors is proposed for dispersive analysis of cell suspensions. The device makes it possible to do a wide range express analysis of suspensions (0.1-600 microns) according to any required number of classes (from 2 to 2(10)). The results of dispersion analysis of the yeast cells of Saccharomyces aragilis crop are given (according to the 16 classes).

[The determination of optimal regimens for producing Klebsiella antigenic erythrocytic immunoreagents]. / Opredelenie optimal'nykh rezhimov polucheniia klebsielleznykh éritrotsitarnykh antigennykh immunoreagentov.

The optimum conditions for obtaining sensitive K- and D-serovar-specific Klebsiella antigenic erythrocyte diagnosticum have been established. The methods of choice are sensitization with lipopolysaccharide antigens at pH 5.0 and in a neutral medium at a temperature of 45 degrees C for an hour. The reagents thus obtained have proved to be specific when tested in the passive hemagglutination test with antisera to Shigella, Salmonella and Escherichia.

[The excretion of Shigella antigens in persons recovering from dysentery]. / Ob ékskretsii antigenov shigell u liudei, perebolevshikh dizenteriei.

Shigella antigens can be detected in the excreta of convalescents after dysentery for a long time. Most frequently these antigens occur in feces, less frequently in urine and rarely in saliva. According to indirect data, S. flexneri 1-6 antigens can be detected in excreta for a longer period after convalescence than S. sonnei antigens. When antigen indication is used for the diagnosis of dysentery and epidemiological analysis is carried out, one should bear in mind the length of the agent persistence in the body, related to Shigella type.

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities.

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.