Study of late toxicity biomarkers of locally advanced head and neck cancer patients treated with radiotherapy plus cisplatin or cetuximab points to the relevance of skin macrophages (TOX-TTCC-2015-01).

PURPOSE: Radiotherapy (RT) with concomitant cisplatin (CRT) or cetuximab (ERT) are accepted treatment options for locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). Long-term adverse events (AEs) have a vast impact on patients' quality of life. This study explored tissue biomarkers which could help predict late toxicity. METHODS/PATIENTS: Single-institution prospective study including patients aged ≥ 18 with histologically confirmed newly diagnosed LA-SCCHN treated with RT and either concomitant cisplatin q3w or weekly cetuximab, according to institutional protocols. All patients underwent pre- and post-treatment skin biopsies of neck regions included in the clinical target volume. Angiogenesis, macrophages, and extracellular matrix (ECM) markers were evaluated by immunohistochemistry (IHC). RESULTS: From April 15, 2016, to December 11, 2017; 31 patients were evaluated [CRT = 12 (38.7%) and ERT = 19 (61.3%)]. 27 patients (87%) had received induction chemotherapy. All patients finished RT as planned. IHC expression of vasculature (CD34) and collagen (Masson's Trichrome) did not differ significantly between and within CRT and ERT arms. Conversely, an increased CD68 and CD163 macrophage infiltration expression was observed after treatment, without significant impact of treatment modality. Patients with higher late toxicity showed lower expression of macrophage markers in pre-treatment samples compared with those with lower late toxicity, with statistically significant differences for CD68. CONCLUSIONS: Angiogenesis and ECM biomarkers did not differ significantly between CRT and ERT. Macrophage markers increased after both treatments and deserve further investigation as predictors of late toxicity in LA-SCCHN patients. [Protocol code: TOX-TTCC-2015-01/Spanish registry of clinical studies (REec): 2015-003012-21/Date of registration: 27/01/2016].

Are there any significant variations in the clinical or histological presentation of lymphoid pathologies over the course of time in Spain?

INTRODUCTION: Little data is available concerning variations in the clinical characteristics of lymphoid neoplasms at presentation. We decided to investigate whether any variations in these characteristics had occurred in Spain during the last few years. MATERIALS AND METHODS: The GOTEL group database is an archive of all new lymphoma cases, regardless of their histological subtype, diagnosed in the hospitals within the group. An analysis was made of all the records between 1 January 1999 and 1 January 2009. Though the number of hospitals submitting data has changed over the course of time, data were provided by 26 hospitals from 16 Spanish provinces. RESULTS: A total of 3651 cases of lymphoma were recorded during this period. Grouped by clinical features, 42.8% (1561 patients) had low-grade lymphoma, 30.4% (1110 patients) intermediate-grade lymphoma and 15.2% (556 patients) Hodgkin's lymphoma; 208 patients had T lymphoma (5.7%), 111 patients high-grade lymphoma (3%) and 105 patients (2.9%) suffered lymphomas that were difficult to classify. A total of 6.3% of the diagnoses (231 patients) were made prior to 1999, 29.5% between 2000 and 2001, 25.7% between 2002 and 2003, 19.7% between 2004 and 2005, 11.2% between 2006 and 2007, and there were 200 entries from 2008 to the close of the study period, corresponding to 1.5% of the complete database. The median age at diagnosis was 60 (range 7-105 years), by percentiles: 25 corresponded to 44 years old, 50 to 60 years old and 75 to 71. Distribution by gender was 53.1% male and 46.9% female. An analysis was made of all the clinical variables collected, comparing their behaviour during the different diagnostic periods. The periods, gender, ECOG, stage, LDH, ß2 microglobulin, Hodgkin's or non- Hodgkin's type neoplasm, B lymphoma vs. Hodgkin's, NK or T, nodal or extra-nodal origin, median age at diagnosis and histological type by region of origin did not show any statistically significant differences in their distribution over the course of time. CONCLUSION: In our experience, there are no significant variations in clinical presentation or histological type in lymphomas diagnosed over the course of time in Spain.

Artificial mixed-linked ß-glucans produced by glycosynthase-catalyzed polymerization: tuning morphology and degree of polymerization.

The glycosynthase derived from Bacillus licheniformis 1,3-1,4-ß-glucanase was able to polymerize glycosyl fluoride donors (G4)(m)G3GαF (m = 0-2, G = Glcß) leading to artificial mixed-linked ß-glucans with regular sequences and variable ß1,3 to ß1,4 linkage ratios. With the E134A glycosynthase mutant, polymers had average molecular masses (M(w)) of 10-15 kDa. Whereas polymer 2 ([4G4G3G](n)) was an amorphous precipitate, the water-insoluble polymers 1 ([4G3G](n)) and 3 ([4G4G4G3G](n)) formed spherulites of 10-20 µm diameter. With the more active E134S glycosynthase mutant, polymerization led to high molecular mass polysaccharides, where M(w) was linearly dependent on enzyme concentration. Remarkably, a homo-polysaccharide [4G4G4G3G](n) with M(w) as high as 30.5 kDa (n ≈ 47) was obtained, which contained a small fraction of products up to 70 kDa, a value that is in the range of the molecular masses of low viscosity cereal 1,3-1,4-ß-glucans, and among the largest products produced by a glycosynthase. Access to a range of novel tailor-made ß-glucans through the glycosynthase technology will allow to evaluate the implications of polysaccharide fine structures in their physicochemical properties and their applications as biomaterials, as well as to provide valuable tools for biochemical characterization of ß-glucan degrading enzymes and binding modules.

Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A beta-glucosidase.

The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(1-->3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(1-->4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.

Glycosynthase activity of Bacillus licheniformis 1,3-1,4-beta-glucanase mutants: specificity, kinetics, and mechanism.

Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2. The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.