Barbara Cardinali

Fibrinogen species as resolved by HPLC-SAXS data processing within the UltraScan Solution Modeler (US-SOMO) enhanced SAS module

The usefulness of a new high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS) data analysis module within the multi-resolution modeling suite US-SOMO is illustrated with size-exclusion small-angle X-ray scattering (SE-SAXS) data of a crude bovine serum albumin sample. The module is then applied to the SE-SAXS study of a human plasma fibrinogen high-molecular-weight fraction presenting severe aggregation problems and a split non-symmetrical main elution peak probably resulting from in-column degradation.

Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200.

Lapatinib concentration in cerebrospinal fluid in two patients with HER2-positive metastatic breast cancer and brain metastases

1. Rusthoven KE, Kavanagh BD, Cardenes H et al. Multi-institutional phase I/II trial of stereotactic body radiation therapy for liver metastases. J Clin Oncol 2009; 27: 1472–1478. 2. Radzikowska E, Glaz P, Roszkowski K. Lung cancer in women: age, smoking, histology, performance status, stage, initial treatment and survival. Populationbased study of 20561 cases. Ann Oncol 2002; 13: 1087–1093. 3. Videtic GM, Reddy CA, Chao ST et al. Gender, race, and survival: a study in nonsmall-cell lung cancer brain metastases patients utilizing the radiation therapy oncology group recursive partitioning analysis classification. Int J Radiat Oncol Phys 2009; 75: 1141–1147. 4. Hendifar A, Yang D, Lenz F et al. Gender disparities in metastatic colorectal cancer survival. Clin Cancer Res 2009; 6391–6397.

Hydrodynamic characterization of recombinant human fibrinogen species

INTRODUCTION Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. METHODS We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. RESULTS We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)(0), s(20,w)(0)) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)(0) and s(20,w)(0) were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. CONCLUSIONS Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bβ residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.

Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress

Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenyl)retinamide (4HPR), the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester), arsenic trioxide (As2O3), and phenylethyl isothiocyanate (PEITC), while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3β (GSK3β). The antioxidant N-acetylcysteine decreased GSK3β phosphorylation and poly(ADP-ribose) polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3β phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1), and transient elevation of intracellular glutathione (GSH) in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3β or transfection with the non-phosphorylatable GSK3β-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3β phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3β phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance.

Response to “A Simplified Implementation of the Bubble Analysis of Biopolymer Networks Pores”

In their comment in this issue of the Biophysical Journal, Munster and Fabry (1) propose a simple and elegant implementation of our bubble method (2) based on the Euclidean distance map (EDM) (3). This EDM bubble method is quite efficient in boosting the performance of our original method because it is fast and at the same time provides the optimal (maximal) coverage of the image, ensuring high accuracy in the parameters recovery (3). Thus, the implementation proposed by Munster and Fabry (1) appears to overcome simultaneously the main two limitations of our original method.

Identification of a new truncated form and deamidation products of fibrinopeptide B released by thrombin from human fibrinogen

Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.

Erratum to: Analysis of in vitro ADCC and clinical response to trastuzumab: Possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines [J Transl Med (2015) 13:324] DOI: 10.1186/s12967-015-0680-0

© 2016 Boero et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Erratum to: J Transl Med (2015) 13:324 DOI 10.1186/s12967‐015‐0680‐0 It has come the publisher’s attention that the original version of this article [1] unfortunately contained an error. In Table 3, first column, the FcγRIIA 131 H>R genotypes were incorrectly labelled. In particular, V/V should have read H/H, V/F should have read H/R and F/F should have read R/R. Please note that this correction does not change the genotype numerical values of FcγRIIA polymorphism. The correct Table 3 has been published as Table 1 in this Erratum. Open Access Journal of Translational Medicine

Pharmacokinetics of Trastuzumab in Haemodialysis

To the Editor: Trastuzumab, a humanized monoclonal antibody which selectively targets the extracellular domains of the human epidermal growth factor receptor 2 (HER2), is used for the treatment of advanced and early breast cancer and advanced gastric cancer overexpressing HER2 (1). Data from the H0649g study with trastuzumab as monotherapy for heavily pretreated women with HER2-positive (HER2+) metastatic breast cancer (MBC) suggest that a dose adjustment is not required in patients with altered renal function (2). However, no detailed studies have been performed on patients with renal impairment and no pharmacokinetic data are available on patients with end-stage renal disease undergoing hemodialysis. Two cases of hemodialyzed patients with HER2+ MBC treated with trastuzumab have been reported in literature (3). Since both patients obtained a clinical response, the authors inferred that a therapeutic concentration of trastuzumab was achieved, but they did not collect data of trastuzumab levels in blood. We report pharmacokinetic data from a hemodialyzed patient with right breast cancer treated with trastuzumab in the adjuvant setting. She was a 64year-old woman affected by Alport-like syndrome with end-stage renal disease undergoing hemodyalisis trice a week. In June 2009, she underwent lumpectomy and axillary lymph node dissection for a 2.4 cm in size, moderately differentiated, hormone receptor negative, HER2+ invasive ductal carcinoma with two lymph nodes positive out of 14 sampled (TNM staging: pT2 N1a M0, IIB). From September 2009, she received six cycles of adjuvant paclitaxel and cyclophosphamide followed by radiotherapy to the right breast. In June 2010, the patient was started on weekly trastuzumab given iv at 4 mg/kg as loading dose and then 2 mg/kg for 1 year. She has not experienced any trastuzumab-related adverse event and at the last follow-up visit in August 2013, she was relapse-free with stable cardiac ejection fraction. Complete pharmacokinetic assessment of trastuzumab was performed on first cycle. Blood samples were drawn at 0 (predose), 1.5 (end of infusion) 3.5, 5.5, 24, 48, 96, and 168 hours after trastuzumab loading dose. Haemodialysis was performed three times a week at 48, 96, and 168 hours after trastuzumab administration. Additional blood samples were obtained 1 hour after the end of each dialysis session (i.e., 52, 100, and 172 hours after trastuzumab administration) to compare preand postdialysis concentration of trastuzumab. Moreover, at each dialysis session, preand postdialyzer samples were collected. Trastuzumab trough concentration was monitored over time, and blood samples were obtained at weeks 4, 7, 12, 18, 24, and 30 before drug administration. Serum was obtained by centrifugation of blood samples and stored at 80°C until analysis. Trastuzumab serum concentrations were evaluated by an enzyme-linked immune-sorbent assay developed and validated for routinely use in our laboratory. The method relies on an antigen peptide corresponding to the portion of HER2 bound by trastuzumab. The antigen is linked to a 96 well plate via the streptavidin/biotin system. The calibration range of the assay was 5–180 lg/mL with a lower limit of quantification of 10 lg/mL. Pharmacokinetic parameters (Cmax, T1/2elim, AUC(0-168h), and Total Body Clearance) were calculated by standard formulas. Trastuzumab serum concentration time curve at the first cycle, pharmacokinetic parameters, and weekly trough levels are shown in Figure 1. The maximum serum concentration after the loading dose administration was 190 mg/L and the weekly trough concentrations ranged from 75 to 163 mg/L. The concentration of trastuzumab did not decrease during hemodialysis and it was similar in the front and rear part of the dialyzer (Table 1). These findings confirm that trastuzumab is not removed from blood through hemodialysis, as it was expected considering the large molecular weight of the drug, which is about 150 kDa. Pharmacokinetic Address correspondence and reprint requests to: Alessandro Inno, MD, PhD, Deparment of Medical Oncology, Ospedale Sacro Cuore Don Calabria, Via don A. Sempreboni 5, 37024 Negrar-Verona, Italy, or e-mail: alessandro.inno@sacrocuore.it