NRG1 fusion-driven tumors: biology, detection, and the therapeutic role of afatinib and other ErbB-targeting agents.

Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to deregulated activity. Fusions involving the neuregulin-1 gene (NRG1) result in ErbB-mediated pathway activation and therefore present a rational candidate for targeted treatment. The most frequently reported NRG1 fusion is CD74-NRG1, which most commonly occurs in patients with invasive mucinous adenocarcinomas (IMAs) of the lung, although several other NRG1 fusion partners have been identified in patients with lung cancer, including ATP1B1, SDC4, and RBPMS. NRG1 fusions are also present in patients with other solid tumors, such as pancreatic ductal adenocarcinoma. In general, NRG1 fusions are rare across different types of cancer, with a reported incidence of <1%, with the notable exception of IMA, which represents ≈2%-10% of lung adenocarcinomas and has a reported incidence of ≈10%-30% for NRG1 fusions. A substantial proportion (≈20%) of NRG1 fusion-positive non-small-cell lung cancer cases are nonmucinous adenocarcinomas. ErbB-targeted treatments, such as afatinib, a pan-ErbB tyrosine kinase inhibitor, are potential therapeutic strategies to address unmet treatment needs in patients harboring NRG1 fusions.

Adenoviral vector expressing murine angiostatin inhibits a model of breast cancer metastatic growth in the lungs of mice.

Angiostatin, an internal fragment of plasminogen, has been shown to inhibit the process of angiogenesis or neovascularization. In this study, we have expressed the cDNA for murine angiostatin under the control of the human cytomegalovirus promoter from a human type-5 adenovirus and shown that this vector produces a protein which retains biological activity. Angiostatin expression was determined by Northern blot analysis and Western immunoblotting. Ad-angiostatin, but not a control vector Ad-dl70, significantly reduced the viability of infected human umbilical cord vein endothelial cells (HUVEC) in vitro. In an in vivo model of basic fibroblast growth factor-induced angiogenesis, Ad-angiostatin (1 x 10(9) pfu) could inhibit endothelial cell migration and the formation of capillaries within a Matrigel plug which had been implanted for one week subcutaneously into C57BL/6 mice. Endothelial cells in these plugs had an altered, rounded, phenotype with dark picnotic nuclei indicative of apoptosis, which was confirmed using transmission electron microscopy. In contrast, endothelial cells from bFGF alone or in combination with the control vector-treated plugs retained the long spindle shape characteristic of endothelial cells. Intranasal delivery of Ad-angiostatin into the lungs of FVB/n mice demonstrated comparable cellular infiltration in the recovered bronchoalveolar lavage fluid with no signs of abnormal pathology as compared to PBS or control vector-treated animals. In a pulmonary metastatic breast cancer model, the delivery of Ad-angiostatin (1 x 10(9) pfu) to the lung significantly delayed tumor growth as measured by the number of visible surface tumor nodules. This study has demonstrated that the specific targeting of tumors to inhibit angiogenesis using an adenovirus expressing angiostatin, may deliver localized concentrations of protein having a greater impact on inhibition of tumor growth.

Combined treatment of a murine breast cancer model with type 5 adenovirus vectors expressing murine angiostatin and IL-12: a role for combined anti-angiogenesis and immunotherapy.

In this study, we used intratumor delivery of adenoviral vectors to induce a selective anti-tumor response by combining the potent angiogenesis inhibitor murine angiostatin (adenovirus (Ad)-angiostatin) with the powerful immune simulator and angiostatic cytokine murine IL-12 (Ad-IL-12). In a murine model of breast carcinoma, intratumor injection of Ad-angiostatin delayed mean tumor growth, as compared with control virus with an initial regression of tumor growth, in 65% of treated animals. However, all treated animals eventually succumbed to the tumors. Mice injected with Ad-IL-12 alone responded with an initial regression in 20% of treated animals, with only 13% developing a total regression. Coinjection of the vectors resulted in 96% of the treated animals developing an initial regression, with 54% undergoing a total regression of the tumor. These mice were resistant to tumor rechallenge and developed a strong CTL response. Frozen tumor sections were stained for microvessel density using an Ab against murine CD31, an endothelial cell marker. Automated image analysis revealed the mean microvessel density following the administration of Ad-angiostatin and Ad-IL-12 alone or in combination was significantly reduced compared with the control-treated tumor. In summary, we have shown that a short-term course of antiangiogenic therapy combined with immunotherapy can effectively shrink a solid tumor and vaccinate the animal against rechallenge. The rationale for this therapy is to limit the tumor size by attacking the vasculature with angiostatin, thereby allowing IL-12 to mount a T cell-specific response against the tumor AG:

Combined CXC chemokine and interleukin-12 gene transfer enhances antitumor immunity.

It has been shown that intratumor administration of an adenovirus vector expressing IL-12 produces a potent T cell-mediated response that leads to significant tumor regression in a murine breast cancer model. IP-10 and MIG are CXC chemokines that recruit mononuclear cells in vivo. In addition to their chemotactic roles, IP-10 and MIG inhibit angiogenesis. We tested whether the addition of IP-10 or MIG may both enhance the antitumor immune response of IL-12 through T cell recruitment and inhibit tumor growth through angiostasis. Adenovirus vectors expressing IP-10 or MIG and/or IL-12 were administered intratumorally in a murine model of mammary adenocarcinoma and fibrosarcoma. Administration of IP-10 or MIG in combination with IL-12 resulted in considerable tumor regression and increased survival time of tumor-bearing animals as compared with IP-10, MIG, IL-12 alone or control-treated animals, with the IP-10 IL-12 combination being most effective. These results suggest augmenting the antitumor immune response and inhibiting tumor angiogenesis with adenoviral vectors expressing IP-10 in combination with IL-12 is a novel way to enhance tumor regression.

Effect of recombinant human interleukin 2 on neutrophil adherence to endothelial cells in vitro.

Human neutrophils were demonstrated to possess interleukin-2 receptor (IL-2R) beta and gamma chains, not alpha chain and the binding of IL-2 to the IL-2R beta chain on neutrophils plays an important regulatory role in neutrophil functions. We have investigated in this study the hypothesis that recombinant human IL-2 (rhIL-2) can directly activate human neutrophils and increase their adherence to human umbilical vein endothelial cells (HUVEC). In an in vitro microtiter adherence assay, rhIL-2 significantly stimulated neutrophil adherence to HUVEC in a dose- and time-dependent manner, rhILI-2 concentration at 2000 u/ml and 2 hour incubation gave the best neutrophil stimulation. Treatment of neutrophils with rhIL-2 increased the expression of adhesion molecule CD18. Pretreatment of the stimulated neutrophils with a blocking monoclonal antibody to CD18 decreased but not completely blocked the adherence of neutrophils to HUVEC. These data suggest than rhIL-2 can directly stimulate and increase neutrophil adherence to HUVEC by enhancing the expression of CD18 and possibly other adhesion molecules on neutrophil surface. This may be a critical step in the early stage of the vascular leak syndrome (VLS) associated with high dose IL-2 therapy.

Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed.