IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

Melanoma cells exhibit variable signal transducer and activator of transcription 1 phosphorylation and a reduced response to IFN-alpha compared with immune effector cells.

PURPOSE: IFN-alpha is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1 activation allows melanoma cells to evade the direct actions of IFN-alpha. EXPERIMENTAL DESIGN: Tyr(701)-phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-alpha-stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC). Expression of other Janus-activated kinase (Jak)-STAT intermediates (STAT1, STAT2, Jak1, tyrosine kinase 2, IFN-alpha receptor, STAT3, and STAT5) was evaluated by flow cytometry, immunoblot, or immunohistochemistry. RESULTS: Significant variability in P-STAT1 was observed in human melanoma cell lines following IFN-alpha treatment (P < 0.05) and IFN-alpha-induced P-STAT1 correlated with the antiproliferative effects of IFN-alpha (P = 0.042). Reduced formation of P-STAT1 was not explained by loss of Jak-STAT proteins or enhanced STAT5 signaling as reported previously. Basal levels of P-STAT3 were inversely correlated with IFN-alpha-induced P-STAT1 in cell lines (P = 0.013). IFN-alpha-induced formation of P-STAT1 was also variable in melanoma cells derived from patient tumors; however, no relationship between P-STAT3 and IFN-alpha-induced P-STAT1 was evident. Because IFN-alpha acts on both tumor and immune cells, we examined the ability of IFN-alpha to induce P-STAT1 in patient-derived melanoma cells and PBMCs. IFN-alpha induced significantly lower levels of P-STAT1 in melanoma cells compared with matched PBMCs (P = 0.046). Melanoma cells and human melanocytes required 10-fold higher IFN-alpha doses to exert P-STAT1 levels comparable with PBMCs. CONCLUSIONS: Melanoma cells are variable in their IFN-alpha responsiveness, and cells of the melanocytic lineage exhibit a lower capacity for IFN-alpha-induced Jak-STAT signaling compared with immune cells.

The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse.

IFN-alpha activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-alpha exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-alpha-stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-alpha treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-alpha. In contrast, STAT1-/- mice could not utilize exogenous IFN-alpha to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1-/- mice was identical regardless of treatment (IFN-alpha or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-alpha. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-alpha in this experimental system.

Neuregulin growth factors and their ErbB receptors form a potential signaling network for schwannoma tumorigenesis.

Sporadic and neurofibromatosis type 2-associated schwannomas contain a glial growth factor (GGF)-like activity that has been hypothesized to promote neoplastic Schwann cell mitogenesis. It is not known whether this GGF-like activity is neuregulin-1 (NRG-1), an epidermal growth factor (EGF)-related molecule that regulates the proliferation, survival, and differentiation of developing Schwann cells, the related factor NRG-2, or another NRG/EGF ligand. We report that neoplastic Schwann cells within schwannomas overexpress multiple alpha and beta transmembrane precursors from the class II and class III NRG-1 subfamilies. NRG-2 alpha and beta transcripts are similarly overexpressed in some tumors. Of the other 8 known NRG/EGF ligands, only heparin-binding EGF, epiregulin, and TGFalpha are detectable in schwannomas. Neoplastic Schwann cells almost uniformly express erbB2 and erbB3, 2 membrane receptor tyrosine kinases mediating NRG-1 and NRG-2 action. Expression of the NRG receptor erbB4 and EGF receptor is also evident in schwannomas, but is more limited, occurring in only a subset of these tumors. ErbB2, the preferred dimerization partner for all erbB kinases, is constitutively phosphorylated in schwannomas. These observations suggest that autocrine, paracrine, and/or juxtacrine NRG-1/NRG-2 signaling promotes schwannoma pathogenesis and that this signaling pathway may be an important therapeutic target in schwannomas.

Interferon alpha and CPG oligodeoxynucleotides elicit additive immunostimulatory and antitumor effects.

BACKGROUND: We hypothesized that interferon alpha (IFN-alpha) and unmethylated cytosine-phosphothioate-guanine (CpG)-rich oligodexoynucleotides (CpG ODNs) would elicit potent antitumor activity due to the ability of this treatment combination to activate complimentary signal transduction intermediates. METHODS: Peripheral blood mononuclear cells treated with CpG ODNs, IFN-alpha, or both agents combined were evaluated for cytotoxicity against human melanoma cells, Jak-STAT signal transduction by flow cytometry, and ISG-15 gene expression by real-time polymerase chain reaction. The effects of CpG ODNs and IFN-alpha were evaluated in murine models of melanoma in wild-type, IFN-gamma-deficient, and STAT1-deficient mice. Negative controls in all experiments included treatment with control ODN or phosphate-buffered saline. RESULTS: Treatment of peripheral blood mononuclear cells with a combination of CpG ODNs and IFN-alpha resulted in enhanced cytotoxicity, activation of natural killer cells, IFN-alpha-induced STAT1 phosphorylation, and transcription levels of ISG-15. These immunostimulatory effects of CpG ODNs were associated with increased expression of STAT1 and STAT2 proteins. Administration of CpG ODNs plus IFN-alpha elicited superior antitumor activity in a murine model of B16 melanoma compared with either agent alone. The antitumor properties of CpG ODNs were dependent on STAT1-mediated signal transduction within the host but independent of endogenously produced IFN-gamma. CONCLUSIONS: CpG ODNs represent potent immune stimulants that elicit antitumor effects through STAT1-mediated signal transduction.

Neuregulin-1 and erbB4 immunoreactivity is associated with neuritic plaques in Alzheimer disease brain and in a transgenic model of Alzheimer disease.

Neuregulin-1 (NRG-1) regulates developmental neuronal survival and synaptogenesis, astrocytic differentiation, and microglial activation. Given these NRG-1 actions, we hypothesized that the synaptic loss, gliosis, inflammation, and neuronal death occurring in Alzheimer disease (AD) is associated with altered expression of NRG-1 and its receptors (the erbB2, erbB3, and erbB4 membrane tyrosine kinases). We examined the expression and distribution of NRG-1 and the erbB kinases in the hippocampus of AD patients and cognitively normal controls and in transgenic mice that coexpress AD-associated mutations of the beta amyloid precursor protein (APP(K670N,M671L)) and presenilin-1 (PS1(M146L)). In the hippocampi of both control humans and wild type mice, NRG-1 and the 3 erbB receptors are expressed in distinct cellular compartments of hippocampal neurons. All 4 molecules are associated with neuronal cell bodies, but only NRG-1, erbB2, and erbB4 are present in synapse-rich regions. In AD and in the doubly transgenic mouse, erbB4 is expressed by reactive astrocytes and microglia surrounding neuritic plaques. In AD brains, microglia and, to a lesser extent, dystrophic neurites, also upregulate NRG-1 in neuritic plaques, suggesting that autocrine and/or paracrine interactions regulate NRG-1 action within these lesions. NRG-1 and erbB4, as well as erbB2, are similarly associated with neuritic plaques in the doubly transgenic mice. Thus, in AD the hippocampal distribution of NRG-1 and erbB4 is altered. The similarities between the alterations in the expression of NRG-1 and its receptors in human AD and in APP(K670N;M671L)/PS1(M146L) mutant mice suggests that this animal model may be very informative in deciphering the potential role of these molecules in AD.

Preparation, biodistribution and neurotoxicity of liposomal cisplatin following convection enhanced delivery in normal and F98 glioma bearing rats.

The purpose of this study was to evaluate two novel liposomal formulations of cisplatin as potential therapeutic agents for treatment of the F98 rat glioma. The first was a commercially produced agent, Lipoplatin™, which currently is in a Phase III clinical trial for treatment of non-small cell lung cancer (NSCLC). The second, produced in our laboratory, was based on the ability of cisplatin to form coordination complexes with lipid cholesteryl hemisuccinate (CHEMS). The in vitro tumoricidal activity of the former previously has been described in detail by other investigators. The CHEMS liposomal formulation had a Pt loading efficiency of 25% and showed more potent in vitro cytotoxicity against F98 glioma cells than free cisplatin at 24 h. In vivo CHEMS liposomes showed high retention at 24 h after intracerebral (i.c.) convection enhanced delivery (CED) to F98 glioma bearing rats. Neurotoxicologic studies were carried out in non-tumor bearing Fischer rats following i.c. CED of Lipoplatin™ or CHEMS liposomes or their "hollow" counterparts. Unexpectedly, Lipoplatin™ was highly neurotoxic when given i.c. by CED and resulted in death immediately following or within a few days after administration. Similarly "hollow" Lipoplatin™ liposomes showed similar neurotoxicity indicating that this was due to the liposomes themselves rather than the cisplatin. This was particularly surprising since Lipoplatin™ has been well tolerated when administered intravenously. In contrast, CHEMS liposomes and their "hollow" counterparts were clinically well tolerated. However, a variety of dose dependent neuropathologic changes from none to severe were seen at either 10 or 14 d following their administration. These findings suggest that further refinements in the design and formulation of cisplatin containing liposomes will be required before they can be administered i.c. by CED for the treatment of brain tumors and that a formulation that may be safe when given systemically may be highly neurotoxic when administered directly into the brain.

Myeloid-derived suppressor cell inhibition of the IFN response in tumor-bearing mice.

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1(+)CD11b(+) MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr(701) in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4(+) and CD8(+) T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide in C26-bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN response as compared with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells.

Pathologic complete response of a solitary melanoma brain metastasis after local ablative radiation therapy: case report.

A 73-year-old female with malignant melanoma metastatic to her left frontal lobe status post-gross total resection of the metastasis, whole brain radiotherapy, and Gamma Knife-based stereotactic radiosurgery for local recurrence presented with an area of increasing enhancement on follow-up magnetic resonance imaging (MRI) and hypermetabolic lesions on 18-fluorodeoxyglucose positron emission tomography/computerized tomography (18FDG PET/CT) of the brain suspicious for tumor recurrence. Surgical resection of the lesion was performed showing radiation necrosis with no evidence of tumor. The patient was alive 1 year after her second craniotomy. This case illustrates that despite being perceived as a radioresistant histology, complete local eradication of melanoma is possible with ablative dose regimens. Prolonged survival is possible in patients with limited metastatic melanoma if local tumor control is achieved.

Advances in oncolytic virus therapy for glioma.

The World Health Organization grossly classifies the various types of astrocytomas using a grade system with grade IV gliomas having the worst prognosis. Oncolytic virus therapy is a novel treatment option for GBM patients. Several patents describe various oncolytic viruses used in preclinical and clinical trials to evaluate safety and efficacy. These viruses are natural or genetically engineered from different viruses such as HSV-1, Adenovirus, Reovirus, and New Castle Disease Virus. While several anecdotal studies have indicated therapeutic advantage, recent clinical trials have revealed the safety of their usage, but demonstration of significant efficacy remains to be established. Oncolytic viruses are being redesigned with an interest in combating the tumor microenvironment in addition to defeating the cancerous cells. Several patents describe the inclusion of tumor microenvironment modulating genes within the viral backbone and in particular those which attack the tumor angiotome. The very innovative approaches being used to improve therapeutic efficacy include: design of viruses which can express cytokines to activate a systemic antitumor immune response, inclusion of angiostatic genes to combat tumor vasculature, and also enzymes capable of digesting tumor extra cellular matrix (ECM) to enhance viral spread through solid tumors. As increasingly more novel viruses are being tested and patented, the future battle against glioma looks promising.

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis.

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-alpha was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-alpha for malignant melanoma.

Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy.

Interferon-alpha (IFN-alpha) is used as an adjuvant therapy in patients with malignant melanoma and who have undergone surgical resection of high-risk lesions. Defective expression or activation of STAT1 or STAT2 has been shown to correlate with IFN-alpha or resistance in vitro; however, recent data from our laboratory suggest that the anti-tumor effects of IFN-alpha are dependent on STAT1 signaling within host immune cells. We measured STAT1 and STAT2 expression in 28 melanoma biopsies (8 cutaneous lesions; 1 lung metastasis; 19 nodal metastases) obtained from patients prior to the initiation of adjuvant IFN-alpha therapy. Disease recurrence following IFN-alpha treatment did not correlate with the staining intensity of either STAT1 (P = 0.61) or STAT2 (P = 0.52). Tumors with minimal STAT1 or STAT2 expression (< 20% positive) were present in four patients with tumor-positive lymph nodes, who exhibited prolonged relapse-free survival (> 44 months) following adjuvant therapy. Conversely, high levels of STAT1 were present in a patient who recurred during the course of IFN-alpha therapy. A case study of one patient who experienced recurrent disease during IFN-alpha treatment revealed that STAT1 levels were greater in the recurrent tumor when compared to the original lesion. These studies provide direct evidence to suggest that levels of STAT1 and STAT2 within the tumor do not influence a patient's response to adjuvant IFN-alpha.