Concomitant CIS on TURBT does not impact oncological outcomes in patients treated with neoadjuvant or induction chemotherapy followed by radical cystectomy.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle invasive bladder cancer improves all-cause and cancer specific survival. We aimed to evaluate whether the detection of carcinoma in situ (CIS) at the time of initial transurethral resection of bladder tumor (TURBT) has an oncological impact on the response to NAC prior to radical cystectomy. PATIENTS AND METHODS: Patients were identified retrospectively from 19 centers who received at least three cycles of NAC or induction chemotherapy for cT2-T4aN0-3M0 urothelial carcinoma of the bladder followed by radical cystectomy between 2000 and 2013. The primary and secondary outcomes were pathological response and overall survival, respectively. Multivariable analysis was performed to determine the independent predictive value of CIS on these outcomes. RESULTS: Of 1213 patients included in the analysis, 21.8% had concomitant CIS. Baseline clinical and pathologic characteristics of the 'CIS' versus 'no-CIS' groups were similar. The pathological response did not differ between the two arms when response was defined as pT0N0 (17.9% with CIS vs 21.9% without CIS; p = 0.16) which may indicate that patients with CIS may be less sensitive to NAC or ≤ pT1N0 (42.8% with CIS vs 37.8% without CIS; p = 0.15). On Cox regression model for overall survival for the cN0 cohort, the presence of CIS was not associated with survival (HR 0.86 (95% CI 0.63-1.18; p = 0.35). The presence of LVI (HR 1.41, 95% CI 1.01-1.96; p = 0.04), hydronephrosis (HR 1.63, 95% CI 1.23-2.16; p = 0.001) and use of chemotherapy other than ddMVAC (HR 0.57, 95% CI 0.34-0.94; p = 0.03) were associated with shorter overall survival. For the whole cohort, the presence of CIS was also not associated with survival (HR 1.05 (95% CI 0.82-1.35; p = 0.70). CONCLUSION: In this multicenter, real-world cohort, CIS status at TURBT did not affect pathologic response to neoadjuvant or induction chemotherapy. This study is limited by its retrospective nature as well as variability in chemotherapy regimens and surveillance regimens.

Nomogram Predicting Bladder Cancer-specific Mortality After Neoadjuvant Chemotherapy and Radical Cystectomy for Muscle-invasive Bladder Cancer: Results of an International Consortium.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) is associated with improved overall and cancer-specific survival. The post-NAC pathological stage has previously been reported to be a major determinant of outcome. OBJECTIVE: To develop a postoperative nomogram for survival based on pathological and clinical parameters from an international consortium. DESIGN, SETTING, AND PARTICIPANTS: Between 2000 and 2015, 1866 patients with MIBC were treated at 19 institutions in the USA, Canada, and Europe. Analysis was limited to 640 patients with adequate follow-up who had received three or more cycles of NAC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A nomogram for bladder cancer-specific mortality (BCSM) was developed by multivariable Cox regression analysis. Decision curve analysis was used to assess the model's clinical utility. RESULTS AND LIMITATIONS: A total of 640 patients were identified. Downstaging to non-MIBC (ypT1, ypTa, and ypTis) occurred in 271 patients (42 %), and 113 (17 %) achieved a complete response (ypT0N0). The 5-yr BCSM was 47.2 % (95 % confidence interval [CI]: 41.2-52.6 %). On multivariable analysis, covariates with a statistically significant association with BCSM were lymph node metastasis (hazard ratio [HR] 1.90 [95% CI: 1.4-2.6]; p < 0.001), positive surgical margins (HR 2.01 [95 % CI: 1.3-2.9]; p < 0.001), and pathological stage (with ypT0/Tis/Ta/T1 as reference: ypT2 [HR 2.77 {95 % CI: 1.7-4.6}; p < 0.001] and ypT3-4 [HR 5.9 {95 % CI: 3.8-9.3}; p < 0.001]). The area under the curve of the model predicting 5-yr BCSM after cross validation with 300 bootstraps was 75.4 % (95 % CI: 68.1-82.6 %). Decision curve analyses showed a modest net benefit for the use of the BCSM nomogram in the current cohort compared with the use of American Joint Committee on Cancer staging alone. Limitations include the retrospective study design and the lack of central pathology. CONCLUSIONS: We have developed and internally validated a nomogram predicting BCSM after NAC and radical cystectomy for MIBC. The nomogram will be useful for patient counseling and in the identification of patients at high risk for BCSM suitable for enrollment in clinical trials of adjuvant therapy. PATIENT SUMMARY: In this report, we looked at the outcomes of patients with muscle-invasive bladder cancer in a large multi-institutional population. We found that we can accurately predict death after radical surgical treatment in patients treated with chemotherapy before surgery. We conclude that the pathological report provides key factors for determining survival probability.

Isomerization and formulation stability of the vaccine adjuvant QS-21.

The stability of the immunologic adjuvant QS-21 (Cambridge Biotech Corp.) was optimized for use in the MN rgp120 HIV-1 subunit vaccine. QS-21, a saponin purified by reversed phase HPLC from an extract of the bark of the Quillaja saponaria Molina tree, consisted initially of one species (QS-21A), but converted to two species, QS-21A and QS-21B, in aqueous solution. NMR studies indicated that the two species are structural isomers and that isomerization occurs by intramolecular trans-esterification of the fatty acid moiety between the 3- and 4-hydroxyl groups of the fucose ring (Jacobsen et al. Carbohydr. Res., in press). Both isomers were adjuvant active. Storage of QS-21 in aqueous solution resulted in the interconversion between these isomer forms, as well as the slow formation of degradation products due to ester hydrolysis. The critical micellar concentration of QS-21 in succinate buffer was measured by a fluorescent probe method to be 51 +/- 9 micrograms/mL. Studies were performed at different concentrations of QS-21 to assess the influence of micelle formation on stability. These experiments indicated that QS-21 is more stable in the micellar form, presumably because the most labile ester bond linking the fatty acid moiety to fucose is constrained or buried in the hydrophobic micellar environment. The pH of maximum stability was pH 5.5, the pH for minimum degradation of most esters. The final formulation, 500 micrograms/mL QS-21 in 20 mM sodium succinate, 150 mM NaCl, pH 5.5, provided a shelf-life of greater than 2 years.

Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy.

The saponin QS-21, derived from the bark of the Quillaja saponaria Molina tree, has shown great potential as an adjuvant with a number of vaccines. Kinetic studies carried out to establish the stability of vaccine formulations show that commercially supplied QS-21 (primarily QS-21A) is converted slowly at pH 5.5, and rapidly at higher pH, to an equilibrium mixture of two regioisomers, QS-21A and QS-21B, in a ratio of 20:1. NMR studies show that QS-21A and QS-21B differ only in the point of attachment of the fatty acyl moiety to the fucose sugar ring. The major isomer, QS-21A, has the fatty acyl portion attached at the 4-hydroxyl group whereas the minor isomer, QS-21B, has the fatty acyl portion attached at the 3-hydroxyl group. The isomerization most likely involves ionization of the 3-hydroxy group and intramolecular acyl transfer from the 4-hydroxy group. The relative stereochemistry of the triterpene and the sugar anomeric centers is also established by NMR methods.

A thioamide substrate of carboxypeptidase A.

Carbobenzoxythioglycyl-L-phenylalanine [CbzNHCH2C(==S)Phe, Z-Glys-Phe] was synthesized as thioamide analogue of Z-Gly-Phe, a known substrate of carboxypeptidase A (CPA). By use of a ninhydrin-based assay and Z-Gly-Gly-Phe as the substrate, Z-Glys-Phe was shown to be a weak competitive inhibitor of CPA (Ki = 1.4 mM). The L isomer (but not the D) of Z-Glys-Phe proved to be a substrate for CPA (Km = 1.1 mM and kcat = 5.3 s-1 at pH 7.5), binding with comparable affinity to, but hydrolyzing at 10% the rate of, the oxo analogue Z-Gly-Phe. The CPA-catalyzed hydrolysis of Z-Glys-Phe was shown to involve only C-N bond cleavage, to give carbobenzoxythioglycine and phenylalanine.

O-Linked glycopeptides retain helicity in water.

A 17-residue O-linked glycopeptide model incorporating a central alpha-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.

4-alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines.

Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine metabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4-2H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4-2H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms.

Antioxidant chemistry of green tea catechins. Identification of products of the reaction of (-)-epigallocatechin gallate with peroxyl radicals.

(-)-Epigallocatechin gallate (EGCG), isolated from green tea, displays antioxidant properties and is thought to act as an antioxidant in biological systems. However, the specific mechanisms of its antioxidant actions remain unclear. In this study, we have isolated and identified for the first time two reaction products of EGCG derived from its reaction with peroxyl radicals generated by thermolysis of the initiator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). The products include a seven-membered B-ring anhydride and a novel dimer. The identification of these products provides the first unambiguous proof that the principal site of antioxidant reactions on the EGCG molecule is the trihydroxyphenyl B ring, rather than the 3-galloyl moiety. In contrast to phenoxyl radicals from simple phenolic antioxidants, an initially formed EGCG phenoxyl radical apparently does not form stable addition products with AMVN-derived peroxyl radicals. Characteristic reaction products may provide novel markers for EGCG antioxidant reactions in living systems.

High-resolution solution structure of the EGF-like domain of heregulin-alpha.

The solution structure of the 63-residue heregulin-alpha (HRG-alpha) epidermal growth factor (EGF)-like domain, corresponding to residues 177-239 of HRG-alpha, has been determined to high resolution using data from two-dimensional and three-dimensional homo- and heteronuclear NMR spectroscopy. The structure is based on a total of 887 internuclear distance and dihedral restraints derived from data obtained using unlabeled and uniformly 15N-labeled protein samples, at pH 4.5, 20 degrees C. A total of 20 structures were calculated using a hybrid distance geometry-simulated annealing approach with the program DGII, followed by restrained molecular dynamics using the program DISCOVER. The average maximum violations are 0.12 +/- 0.01 angstroms and 1.4 +/- 0.3 degrees for distance and dihedral restraints, respectively. The backbone (N,C(alpha),C) atomic rms distribution about the mean coordinates for residues 3-23 and 31-49 is 0.29 +/- 0/07 angstroms. The N-and C-terminal residues (1-2 and 50-63) and 24-30 are disordered. Comparison of the HRG-alpha EGF-like domain structure with the previously determined structure of human EGF [Hommel et al. (1992) J. Mol. Biol. 227, 271-282] reveals a high degree of structural similarity; excluding the N-terminal region (residues 1-13), the disordered phi-loop region (residues 24-30) that contains a three-residue insertion in HRG-alpha relative to hEGF, and the disordered C-terminal region (residues 50-63), the C(alpha) alignment between the HRG-alpha and hEGF minimized mean structures has a rms difference of approximately 1 angstrom. In HRG-alpha the N-terminal residues 2-6 form a well-defined beta strand rather than being disordered as found for hEGF. This structural difference correlates with functional data which suggest that the N-terminal region of the HRG-alpha EGF-like domain is responsible for the observed receptor specificity differences between HRG-alpha and EGF.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.