Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients’ peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by 51Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

Fibrinogen is a large heterogeneous aggregation/degradation-prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a high-molecular-weight fraction was analyzed by size-exclusion high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of theUltraScan Solution Modeler(US-SOMO; http://somo.uthscsa.edu/), an open-source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non-baseline-resolved HPLC-SAXS elution peaks. It was thus possible to resolve at least two species co-eluting under the fibrinogen main monomer peak, probably resulting from in-column degradation, and two others under an oligomers peak. The overall and cross-sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi-automated procedures available within theUS-SOMOSAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This newUS-SOMOversion additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions ofP(r), and an advanced module for the comparison of primaryI(q)versus qdata with model curves computed from atomic level structures or bead models. It should be of great help in multi-resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data.

Great improvement has been made in the diagnosis and therapy of breast cancer patients. However, the identification of biomarkers for early diagnosis, prognosis, therapy assessment and monitoring, including drug resistance and the early detection of micro-metastases, is still lacking. Recently, circulating microRNAs (miRNAs), circulating freely in the blood stream or entrapped in extracellular vesicles (EVs), have been shown to have a potential diagnostic, prognostic or predictive power. In this review, recent findings are summarized, both at a preclinical and clinical level, related to miRNA applicability in the context of breast cancer. Different aspects, including clinical and technical challenges, are discussed, describing the potentialities of miRNA use in breast cancer. Even though more methodological standardized studies conducted in larger and selected patient cohorts are needed to support the effective clinical utility of miRNA as biomarkers, they could represent novel and accessible tools to be transferred into clinical practice.