Does Immunohistochemistry Represent a Robust Alternative Technique in Determining Drugable Predictive Gene Alterations in Non-Small Cell Lung Cancer?

Immunohistochemistry (IHC) is a widely-tested, low-cost and rapid ancillary technique available in all laboratories of pathology. This method is generally used for diagnostic purposes, but several studies have investigated the sensitivity and specificity of different immunohistochemical antibodies as a surrogate test in the determination of predictive biomarkers in non-small cell lung cancer (NSCLC), particularly for Epidermal Growth Factor Receptor (EGFR) gene mutations, Anaplastic Lymphoma Kinase (ALK) gene and ROS1 rearrangements. In this review, a critical examination of the works comparing the consistency of IHC expression and conventional molecular techniques to identify genetic alterations with predictive value in NSCLC is discussed. Summarizing, data on sensitivity and specificity of antibodies against ALK and ROS1 are very consistent and time has come to trust in IHC at least as a cost-effective screening tool to identify patients with rearranged tumors in clinical practice. On the other hand, mutant-specific antibodies against EGFR demonstrate a good specificity but a lowto- fair sensitivity, raising some cautions on their employment as robust predictive biomarkers. A brief comment on preliminary experiences with antibodies against BRAF, RET, HER2 and c-MET is also included.

A role for WNT1-inducible signaling protein-1 in airway remodeling in a rat asthma model.

Over-expression of WISP1 has been described in multi-organ fibrosis and tissue remodeling. Moreover, it has recently been found that polymorphism of WISP1 gene is related with the change of lung function in asthmatic subjects. Therefore, we hypothesized that WISP1 might be closely linked to occurrence and development of asthmatic airway remodeling. Aim of this study was to examine the roles of WISP1 in an asthmatic model with airway remodeling and assess the specific effects of WISP1 on human lung fibroblast in vitro. Animal models were developed by challenged with ovalbumin. The levels of WISP1 expression in animal models were assessed by real-time PCR and immunohistochemistry. To examine the specific effects of WISP1 on airway remodeling, WISP1 was depleted by neutralizing α-WISP1 antibodies in vivo. Moreover, human lung fibroblast (HFL-1) was challenged with WISP1 in the presence and absence of SH-5 in vitro. Our study showed that OVA exposure increased the levels of WISP1 expression in a rat asthma model. WISP1 depletion could partially inhibit OVA-induced airway remodeling. In vitro, WISP1-treated HFL-1 cells presented abnormal proliferation and over-expression of Col1a1 and Fn1. However, WISP1-induced collagen release from HFL-1 cells could be attenuated by pretreatment with an Akt inhibitor. Moreover, the levels of p-Akt and p-GSK-3ß in WISP1-treated HFL-1 cells were also significantly elevated. In summary, WISP1 might initiate and perpetuate the pathological remodeling of asthma by inducing fibroblast proliferation and ECM deposition. The specific effects of WISP1 were likely due to activation of pulmonary Akt/GSK-3ß signaling.

Endocytic internalization of adenovirus, nonspecific phagocytosis, and cytoskeletal organization are coordinately regulated in alveolar macrophages by GM-CSF and PU.1.

GM-CSF gene-targeted (GM(-/-)) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM(-/-) mice compared to wild-type (GM(+/+)) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM(-/-) mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM(-/-) mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM(+/+) AM cell line. Phagocytosis of 4- micro m latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM(-/-) AMs restored phagocytosis of 4- micro m beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin alpha(V) and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110gamma isoform, important in macrophage phagocytic function, was absent in GM(-/-) AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small ( approximately 100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.

Assessment of the anti-c-kit monoclonal antibody YB5.B8 in affinity magnetic enrichment of human lung mast cells.

The monoclonal antibody, YB5.B8 binds to the second domain of the c-kit proto-oncogene product on human mast cells, a receptor associated with tyrosine kinase activity. This molecule is involved with cell proliferation, maturation and viability as well as cell activation and its natural ligand is stem cell factor (SCF). We have used this antibody coupled to Dynabeads to perform positive affinity enrichment of human lung mast cells. This procedure results in enrichment of mast cells from 2.6 +/- 0.3% to 85.0 +/- 1.6% purity (n = 29) with yields of 41.9 +/- 3.7% (n = 29). As YB5.B8 interacts with the same receptor domain as does SCF, it is important to demonstrate that this procedure does not modify mast cell function. Incubation of mast cells with 1-5000 ng/ml YB5.B8 for 30 min neither induced histamine release nor modulated histamine release induced by anti-IgE. Furthermore, incubation with YB5.B8 did not alter prolonged culture with SCF. Examination of cells enriched using YB5.B8 showed that they had a normal histamine content (3.8 +/- 0.3 pg/cell compared with 3.9 +/- 0.7 pg/cell unpurified, n = 20) and had unchanged behaviour in both histamine secretion and cell survival studies. These studies indicate that YB5.B8 does not influence mast cell function and thus its use in magnetic affinity purification procedures offers a simple and effective method for enriching human mast cell preparations.