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Because epigenetic inhibitors can reduce cancer cell proliferation, we tested the hypothesis that concurrent inhibition of histone acetylation and DNA methylation could synergistically reduce the viability of small cell lung cancer (SCLC) cells. Sub-IC50 concentrations of the DNA methyltransferase (DNMT) inhibitor decitabine (5-AZA-dC) and the histone deacetylase (HDAC) inhibitors (LBH589 or MGCD0103) synergistically reduced the proliferation of five of nine SCLC cell lines. Loss of viability of sensitive SCLC cells did not correlate with the inhibition of either DNMT1 or HDACs, suggesting nonepigenetic mechanisms for synergy between these two classes of epigenetic modulators. Because combinations of 5-AZA-dC and HDAC inhibitors had marginal effects on the apoptosis index, Comet assay was undertaken to assess DNA damage. MGCD0103 and 5AZA-dC cotreatment augmented DNA damage in SCLC cells, resulting in increased tail length and moment in Comet assays by 24 hours in sensitive cell lines (P < 0.01). Consistent with augmented DNA damage, combination of a DNMT and HDAC inhibitor markedly increased the levels of phospho-H2A.X in sensitive cells but not in resistant ones. Comparison of basal gene expression between resistant and sensitive cells identified markedly higher basal expression of IFN-stimulated genes in the resistant cell lines, suggesting that IFN-stimulated gene expression may determine SCLC cell sensitivity to epigenetic modulators or other DNA damaging agents. Mol Cancer Ther; 9(8); 2309–21. ©2010 AACR.

Supplementary Data from Combinations of DNA Methyltransferase and Histone Deacetylase Inhibitors Induce DNA Damage in Small Cell Lung Cancer Cells: Correlation of Resistance with IFN-Stimulated Gene Expression

Since an all-inclusive review of the interferon system is beyond the scope of this chapter, its purpose will be to present a general overview of interferons and the induction processes. Minus-stranded RNA viruses possess a transcriptase that generates complementary RNA in the absence of RNA replication; thereby enabling the production of double-stranded RNA and subsequent interferon induction despite UV inactivation. In studies comparing interferon induction by polynucleotides and viruses, different kinetics of induction have been observed. In addition to evidence on the molecular level in support of this hypothesis, it is attractive in that it better explains interferon induction by metabolic inhibitors than does the double-stranded RNA hypothesis. To dissect the importance of these promotor regions to normal interferon induction, genetic hybrids have been constructed. Mechanisms of a induction of interferons fall into two categories, classical and an immune induction.

Over the past decade, preclinical and clinical research have confirmed the essential role of interferons for effective host immunological responses to malignant cells. Type I interferons (IFNα and IFNβ) directly regulate transcription of >100 downstream genes, which results in a myriad of direct (on cancer cells) and indirect (through immune effector cells and vasculature) effects on the tumour. New insights into endogenous and exogenous activation of type I interferons in the tumour and its microenvironment have given impetus to drug discovery and patient evaluation of interferon-directed strategies. When combined with prior observations or with other effective modalities for cancer treatment, modulation of the interferon system could contribute to further reductions in cancer morbidity and mortality. This Review discusses new interferon-directed therapeutic opportunities, ranging from cyclic dinucleotides to genome methylation inhibitors, angiogenesis inhibitors, chemoradiation, complexes with neoantigen-targeted monoclonal antibodies, combinations with other emerging therapeutic interventions and associations of interferon-stimulated gene expression with patient prognosis - all of which are strategies that have or will soon enter translational clinical evaluation.

We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-kappa B was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of I kappa B alpha, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of I kappa B alpha and inhibition of NF-kappa B DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-alpha-mediated activation of a transfected NF-kappa B-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination.

At times we encounter clinical problems for which there are no directly applicable evidence-based solutions, but we are compelled by circumstances to act. When doing so we rely on related evidence, general principles of best medical practice, and our experience. Each “Current Clinical Practice” feature article in Seminars in Oncology describes such a challenging presentation and offers treatment approaches from selected specialists. We invite readers' comments and questions, which, with your approval, will be published in subsequent issues of the Journal. It is hoped that sharing our views and experiences will better inform our management decisions when we next encounter similar challenging patients. Please send your comments on the articles, your challenging cases, and your treatment successes to me at dr.gjmor ris@gmail.com. I look forward to a lively discussion. Gloria J. Morris, MD, PhD Current Clinical Practice Feature Editor

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.

Background: Circulating tumor cells (CTC) predict overall survival in patients with metastatic prostate cancer. The objective of this study is to measure CTC before radical prostatectomy in intermediate- and high-risk prostate cancer patients. Materials and Methods: The study accrued 12 patients and 10 provided adequate peripheral blood sample. Blood was drawn preoperatively and assayed for CTC using the CellSearch system. Patients were categorized as CTC positive (≥ 1 CTC) or CTC negative (no CTC). Results: Median age was 64.5 years (range 49-77 years), median prostate specific antigen was 7.4 ng/ml (range 5.7-25.7 ng/ml). Seven patients had intermediate-risk and 3 patients had high-risk prostate cancer. One patient was found to be CTC positive. Conclusions: Our pilot study shows that CTC are rare in patients with clinically localized disease despite intermediate- to high-risk features. CTC may not be the optimal marker to predict prognosis or detect residual disease after radical prostatectomy.

We recently identified neuregulin-1 (NRG1) as a novel target of Notch1 required in Notch-dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1-dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1-ERBB3-ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.

Since sodium stibogluconate (SSG) inhibited phosphatases including SHP-1 and augmented anti-tumor actions of IFN-α2b in vitro and in mice, two Phase I trials of SSG/IFN-α2b combination were undertaken to evaluate safety and target inhibition. Escalating doses of SSG (200-1200 mg/m2) and fixed doses of IFN-α2b (3x106 units/m2) with or without chemotherapy (dacarbazine, vinblastine, cisplatin) were evaluated for side effects and impact on SHP-1 phospho-substrates and IFNα-stimulated-genes (ISGs) in peripheral blood in 40 patients with metastatic melanoma, soft tissue sarcomas, gastrointestinal stromal tumors, and breast or colorectal carcinomas who did not have other established treatment options. Common adverse events were bone marrow suppression, fatigue, gastrointestinal upset, and asymptomatic lipase elevation (n=13); the latter was dose related and mostly after 10d of SSG/IFN-α2b in combination. Levels of SHP-1 substrates (pSTAT1, pSTAT3, pLck and pSlp76) were increased (up to 3x) in peripheral blood cells following SSG with no potentiation by combination with IFN-α2b. Representative ISGs in peripheral blood were induced after IFN-α2b at 4 and 24 hrs with selective modulations by combination. The median time on trials was 2.3 months (10-281d) with no objective regression of disease. Alive at 1y were 17/40 (43%) patients and after 2y were 8/40 (20%) following treatment initiation. These data demonstrate that SSG impacted signal molecules consistent with PTP inhibition and was tolerated in combination with IFN-α2b. Phase II investigations of SSG could safely utilize doses of up to 1200 mg/m2 of SSG for up to 10d alone or in combination with IFN-α2b with or without chemotherapy.

Antibody, small molecule and protein inhibitors of angiogenesis are used in the management of several cancers. These do not specifically target tumor vascularity, and resistance can be problematic. VB-111 is a vascular-targeting agent consisting of a non-replicating adenovirus vector with a pre-proendothelin-1 promoter that encodes an apoptotic receptor.The rationale and design of VB-111, its mechanism of action, and preclinical studies examining antitumor activity, toxicology and pharmacodynamics are reviewed. Phase I and Phase II clinical trials are also reviewed.VB-111 is a vascular-targeting gene therapeutic that is both tissue- and condition-specific, with effects limited to endothelial cells undergoing angiogenesis. Systemic administration produces selective destruction of tumor vascularity. Synergistic antitumor activity can be observed when combined with chemotherapy. VB-111 has been found to be safe and well tolerated in a Phase I clinical trial in patients with advanced solid tumors. Phase II clinical trials are in progress. VB-111 is novel agent for cancer that may have application as monotherapy and in combination with other therapies.

BACKGROUND: Patients with advanced soft tissue sarcomas (STS) have limited therapeutic options. Sorafenib (BAY 43-9006) is a multitargeted tyrosine kinase inhibitor of raf, vascular endothelial growth factor receptors 1 (VEGFR1) through 3, platelet-derived growth factor B, fms-like tyrosine kinase 3, and c-kit, and some of these may be relevant in STS. METHODS: The authors tested sorafenib at a dose of 400 mg twice daily in patients with advanced vascular sarcoma (VS), high-grade liposarcomas, and leiomyosarcomas who had received 0 or 1 previous regimens for advanced disease. RESULTS: Fifty-one patients were accrued to the study, and 37 were evaluable for toxicity and response. There were no unexpected side effects and no confirmed responses. The median progression-free survival was 3 months, and the median overall survival was 17 months. Six of 8 patients in the VS cohort had prolonged clinical benefit (stable disease or better), resulting in a median progression-free survival of 5 months compared with 2 to 3 months for the patients who had liposarcoma and leiomyosarcomas. CONCLUSIONS: Sorafenib at the dose and schedule studied did not result in any responses in the VS, liposarcoma, or leiomyosarcoma cohort according to Response Evaluation Criteria in Solid Tumors. Cancer 2012;. © 2011 American Cancer Society.

To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function. Peripheral blood mononuclear cells (PBMCs) from healthy control subjects and from patients with newly diagnosed glioma were stimulated with anti-CD3/anti-CD28 and T cells assessed for intracellular expression of interferon (IFN)-γ. Antibody staining of PBMCs from glioma patients and healthy donors (CD33, HLADR, CD15, and CD14) followed by 4-color flow cytometry analysis-defined MDSC levels in the peripheral blood. To assess the role of MDSCs in suppressing T cell IFNγ production, PBMCs were depleted of MDSCs using anti-CD33 and anti-CD15 antibody-coated beads prior to T cell stimulation. Enzyme-linked immunosorbent assays were used to assess plasma arginase activity and the level of granulocyte colony-stimulating factor (G-CSF). Patients with glioblastoma have increased MDSC counts (CD33+HLADR-) in their blood that are composed of neutrophilic (CD15(+);60%), lineage-negative (CD15(-)CD14(-); 31%), and monocytic (CD14(+); 6%) subsets. After stimulation, T cells from patients with glioblastoma had suppressed IFN-γ production when compared with healthy, age-matched donor T cells. Removal of MDSCs from the PBMCs with anti-CD33/CD15-coated beads significantly restored T cell function. Significant increases in arginase activity and G-CSF levels were observed in plasma specimens obtained from patients with glioblastoma. The accumulation of MDSCs in peripheral blood in patients with glioma likely promotes T cell immune suppression that is observed in this patient population. Increased plasma levels of arginase and G-CSF may relate to MDSC suppressor function and MDSC expansion, respectively, in patients with glioma.

Interferon (IFN)-β in preclinical studies, compared to IFN-α2, bound with higher affinity to its receptor, induced to higher levels of IFN-stimulated gene products, induced more apoptosis in melanoma cells, and had antitumor effects against melanoma. A maximally tolerated dose of 12 × 10(6) international units/m(2) after 2 weeks subcutaneously daily with dose escalation to 18 × 10(6) international units/m(2) was thus used in a phase II trial of IFN-β1a in cutaneous metastatic melanoma (n = 17) and uveal melanoma (n = 4). It resulted in expected but reversible drug-related severe (grade 3) adverse events in 13/21 patients; anorexia and fatigue were mostly of mild or moderate severity and infrequently needed dose reduction. Although a single patient had a sustained regression, overall IFN-β1a did not have clinical benefit (response rate10%; median progression-free survival 1.8 months). Effective and potent induction in peripheral blood cells and into serum of products of IFN-stimulated genes such as the pro-apoptotic cytokine, TRAIL, and the immunomodulatory and anti-angiogenic chemokines, CXCL10 and CCL8, confirmed gene regulatory actions. To probe further anti-angiogenic mechanisms, both VEGF-A and CXCL-5 were assessed; compared to before treatment, both proteins decreased. Continued improvements in understanding of antitumor mechanisms will enhance usefulness of IFNs for nodal or distant metastases from melanoma.

Immunotherapeutics have been applied intratumorally to manage accessible lesions and to induce systemic immunity in malignant melanoma. Intratumoral bacillus Calmette-Guérin (BCG) has been used for 40 years, and intratumoral BCG, IL-2, IFN-α and imiquimod are recommended as treatment options for patients with in-transit melanoma metastases. Regression of cutaneous metastases can be achieved. Subcutaneous metastases are more refractory, and regression of uninjected, visceral metastases is infrequent. Other microbial products, cytokines, chemicals, immune cells, antibody and viral and plasmid vectors expressing immunologically active molecules have been tested. Antitumor activity has not been demonstrated to be superior to that of intratumoral BCG. There are few controlled trials, and whether survival is impacted with any approach has not yet been established. The immunotherapeutics applied and the intratumoral administration procedure itself can activate responses that are immune inhibitory. More rigorous clinical testing and improved understanding and modulation of regulatory immune responses are necessary.

Studies of the action of interferon-stimulated genes (ISGs) and their protein products have resulted in fundamental discoveries relevant to translational control, regulation of RNA stability and editing, and protein transport and turnover. Actions of ISGs will remain critical to improved clinical application of agonists and antagonists of the toll-like receptor and the interferon signaling cascades--now 25 years after the U.S. Food and Drug Administration and worldwide regulatory approval of the pharmaceutical product produced by recombinant DNA technology. Because the antiviral and cellular actions of these several hundred genes (what?) and their protein products are now being functionally (how?) further elucidated but have been comprehensively summarized to only limited extents, we have selected some of the most potently induced ISGs for review in this special issue of the Journal of Interferon & Cytokine Research.

Interferons (IFNs) manifest their cellular functions by regulating expression of target genes known collectively as IFN-stimulated genes (ISGs). The repertoires of ISGs vary slightly between cell types, but routinely include a core of common ISGs robustly upregulated in most IFN-treated cells. Here, we review the regulation and cellular functions of 2 related ISGs, ISG12 (IFI27) and G1P3 (ISG 6-16), that are commonly induced by IFNs in most, if not all, IFN-responsive cells. On the basis of sequence similarity, they are grouped together within the newly defined FAM14 family. Emerging data on ISG12 and G1P3 suggest that both are mitochondrial proteins with opposing activities on apoptosis that may influence the innate immune responses of IFNs. The G1P3 gene encodes a low molecular weight mitochondrial protein that may stabilize mitochondrial function and oppose apoptosis. In contrast, ISG12 expression may sensitize cells to apoptotic stimuli via mitochondrial membrane destabilization. On the basis of these results and differences in induction kinetics between ISG12 and G1P3, we have proposed a model for the role of these genes in mediating cellular activity of IFNs.

Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.

Because epigenetic inhibitors can reduce cancer cell proliferation, we tested the hypothesis that concurrent inhibition of histone acetylation and DNA methylation could synergistically reduce the viability of small cell lung cancer (SCLC) cells. Sub-IC50 concentrations of the DNA methyltransferase (DNMT) inhibitor decitabine (5-AZA-dC) and the histone deacetylase (HDAC) inhibitors (LBH589 or MGCD0103) synergistically reduced the proliferation of five of nine SCLC cell lines. Loss of viability of sensitive SCLC cells did not correlate with the inhibition of either DNMT1 or HDACs, suggesting nonepigenetic mechanisms for synergy between these two classes of epigenetic modulators. Because combinations of 5-AZA-dC and HDAC inhibitors had marginal effects on the apoptosis index, Comet assay was undertaken to assess DNA damage. MGCD0103 and 5AZA-dC cotreatment augmented DNA damage in SCLC cells, resulting in increased tail length and moment in Comet assays by 24 hours in sensitive cell lines (P < 0.01). Consistent with augmented DNA damage, combination of a DNMT and HDAC inhibitor markedly increased the levels of phospho-H2A.X in sensitive cells but not in resistant ones. Comparison of basal gene expression between resistant and sensitive cells identified markedly higher basal expression of IFN-stimulated genes in the resistant cell lines, suggesting that IFN-stimulated gene expression may determine SCLC cell sensitivity to epigenetic modulators or other DNA damaging agents. Mol Cancer Ther; 9(8); 2309–21. ©2010 AACR.

Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic, or targeted therapeutics. Being an antiapoptotic mediator of chemoresistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemoresistance and improving therapeutic efficacy. In this work, tyrosine phosphatase inhibitor-3 (TPI-3) was identified as a novel small molecule inhibitor of MKP1 and was capable of sensitizing tumors to bio- and chemotherapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phosphosubstrates in Jurkat cells and induced cell death via apoptosis at nanomolar concentrations. TPI-3 also increased MKP1 phosphosubstrates in WM9 human melanoma cells and synergized with biotherapeutic IFNα2b in the growth inhibition of melanoma cells in vitro (combination index

Purpose To assess potential differences in progression-free or overall survival when imatinib mesylate is administered to patients with incurable gastrointestinal stromal tumors (GIST) at a standard dose (400 mg daily) versus a high dose (400 mg twice daily). Patients and Methods Patients with metastatic or surgically unresectable GIST were eligible for this phase III open-label clinical trial. At registration, patients were randomly assigned to either standard or high-dose imatinib, with close interval follow-up. If objective progression occurred by Response Evaluation Criteria in Solid Tumors, patients on the standard-dose arm could reregister to the trial and receive the high-dose imatinib regimen. Results Seven hundred forty-six patients with advanced GIST from 148 centers across the United States and Canada were enrolled onto this trial in 9 months. With a median follow-up of 4.5 years, median progression-free survival was 18 months for patients on the standard-dose arm, and 20 months for those receiving high-dose imatinib. Median overall survival was 55 and 51 months, respectively. There were no statistically significant differences in objective response rates, progression-free survival, or overall survival. After progression on standard-dose imatinib, 33% of patients who crossed over to the high-dose imatinib regimen achieved either an objective response or stable disease. There were more grade 3, 4, and 5 toxicities noted on the high-dose imatinib arm. Conclusion This trial confirms the effectiveness of imatinib as primary systemic therapy for patients with incurable GIST but did not show any advantage to higher dose treatment. It appears reasonable to initiate therapy with 400 mg daily and to consider dose escalation on progression of disease.

The effectiveness of IFN-alpha2b for human multiple myeloma has been variable. TRAIL has been proposed to mediate IFN-alpha2b apoptosis in myeloma. In this study we assessed the effects of IFN-alpha2b signaling on the apoptotic activity of TRAIL and human myeloma cell survival. While TRAIL was one of the most potently induced proapoptotic genes in myeloma cells following IFN-alpha2b treatment, less than 20% of myeloma cells underwent apoptosis. Thus, we hypothesized that an IFN-stimulated gene (ISG) with prosurvival activity might suppress TRAIL-mediated apoptosis. Consistent with this, IFN-alpha2b stabilized mitochondria and inhibited caspase-3 activation, which antagonized TRAIL-mediated apoptosis and cytotoxicity after 24 hours of cotreatment in cell lines and in fresh myeloma cells, an effect not evident after 72 hours. Induced expression of G1P3, an ISG with largely unknown function, was correlated with the antiapoptotic activity of IFN-alpha2b. Ectopically expressed G1P3 localized to mitochondria and antagonized TRAIL-mediated mitochondrial potential loss, cytochrome c release, and apoptosis, suggesting specificity of G1P3 for the intrinsic apoptosis pathway. Furthermore, RNAi-mediated downregulation of G1P3 restored IFN-alpha2b-induced apoptosis. Our data identify the direct role of a mitochondria-localized prosurvival ISG in antagonizing the effect of TRAIL. Curtailing G1P3-mediated antiapoptotic signals could improve therapies for myeloma or other malignancies.

Increased expression of genes, silenced by methylation of their promoters, could have relevance for increasing effects of not only interferons (IFNs) but also APO2L/TRAIL, cytotoxics and immunotherapeutics for melanoma and other malignancies. A resistant melanoma cell line, A375, lacked APO2L/TRAIL or apoptosis induction by either IFN-alpha2 or IFN-beta. However, apoptosis was induced by IFNs in A375 cells by 5-aza,2'-deoxycytidine (5-Aza-dC), evaluated based upon the postulate that promoter methylation might be silencing pro-apopoptotic IFN-stimulated genes (ISGs). RASSF1A, commonly methylated at high frequency in many tumors including melanoma, which we discovered to be also an IFN-regulated gene, was increased by 5-Aza-dC. RASSF1A was important in enhancing apoptotic effects of not only IFNs and APO2L/TRAIL but also cisplatin. Unraveling epigenetic regulatory mechanisms, as yet only partially identified, will result in new biological insights and improved strategies for therapeutic use of IFNs or ISGs such as APO2L/TRAIL.

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.

Lenalidomide is an immunomodulatory derivative of thalidomide with significantly greater in vitro activity and a different toxicity profile. In preclinical trials it has shown synergy with chemotherapy.Primary objective of this study was to determine the maximum tolerated doses of docetaxel and carboplatin when combined with oral lenalidomide in a standard phase I study design. Between September 2004 and May 2005, 14 patients with pathologically proven solid tumors,or =2 prior chemotherapy regimens, performance status ECOG 0/1, and adequate organ function were enrolled. Dose limiting toxicities (DLT) were defined asor = grade 3 non-hematological, or grade 4 hematological toxicity. No growth factors were used during cycle 1.Three of four patients treated at dose level 1, docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, and lenalidomide 10 mg orally daily on Days 1-14 of a 21 day cycle experienced DLT (grade 3 electrolyte changes in two patients, and grade 4 neutropenia in one patient). Ten patients were treated at dose level -1, docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, and lenalidomide 5 mg orally daily on Days 1-14 of a 21 day cycle with one DLT (Grade 4 neutropenia). There were no treatment-related deaths or irreversible toxicities. Of the 14 response-evaluable patients, five achieved a partial response (5 out of 9 patients with non-small cell lung cancer.Docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, with lenalidomide 5 mg orally daily on Days 1-14 days of a 21 day cycle is the maximum tolerated dose without the use of prophylactic growth factors. This combination is active and further evaluation in a phase II trial is warranted.

Preclinical studies had determined that tamoxifen and interferon-alpha2b (IFN-alpha2b) synergistically inhibited growth of both estrogen-receptor positive and negative murine tumor xenografts and had combined antiangiogenic effects and that tamoxifen potentiated IFN-stimulated gene (ISG) expression. A phase I trial in 26 patients was conducted using the combination to define tolerance and potentiation of ISG expression. IFN- alpha2b at a dose of 3 x 10(6) units/m(2) daily was given subcutaneously (s.c.), and tamoxifen was initiated as a loading dose of 150 mg/m(2) and then 60 mg/m(2) twice daily on day 8. At this initial dose, reduction of dose of IFN- alpha2b was required in 4 of 11 patients, primarily because of fatigue. Another group of patients was treated with an identical tamoxifen dose but with IFN-alpha2b reduced to 2 x 10(6)/m(2) U; this was better tolerated. As the projected serum tamoxifen level to reduplicate preclinical effects was 300 mg/m(2), dose escalation in a third cohort was undertaken; it had to be discontinued secondary to grade III or IV toxicity in 2 of 2 patients. Increases in products of transcriptionally regulated ISGs, beta (2)-microglobulin, neopterin, and ISG15 were assessed. All ISGs increased after IFN-alpha2b, but only ISG15 had a further significant rise after initiation of tamoxifen. Because at doses not limited by unacceptable toxicities, no marked potentiation of ISGs by tamoxifen could be identified, clinical evaluation of the combination was terminated.

Purpose: The presence of ≥5 circulating tumor cells (CTC) in 7.5 mL blood from patients with measurable metastatic breast cancer before and/or after initiation of therapy is associated with shorter progression-free and overall survival. In this report, we compared the use of CTCs to radiology for prediction of overall survival. Experimental Design: One hundred thirty-eight metastatic breast cancer patients had imaging studies done before and a median of 10 weeks after the initiation of therapy. All scans were centrally reviewed by two independent radiologists using WHO criteria to determine radiologic response. CTC counts were determined ∼4 weeks after initiation of therapy. Specimens were analyzed at one of seven laboratories and reviewed by a central laboratory. Results: Interreader variability for radiologic responses and CTC counts were 15.2% and 0.7%, respectively. The median overall survival of 13 (9%) patients with radiologic nonprogression and ≥5 CTCs was significantly shorter than that of the 83 (60%) patients with radiologic nonprogression and Conclusions: Assessment of CTCs is an earlier, more reproducible indication of disease status than current imaging methods. CTCs may be a superior surrogate end point, as they are highly reproducible and correlate better with overall survival than do changes determined by traditional radiology.

A total of 419 patients with progressive liver disease, in nearly all cases metastatic from gastrointestinal primaries, were treated by intrahepatic arterial infusion with 5-FU. Three-fourths of these patients had had prior trials with intravenous 5-FU for 1 or 2 months to several years and had been switched to the infusion upon the development of progression. Catheters were placed percutaneously and the patients infused with 5-FU at a dose of 20 to 30 mg kg/day × 4, then 15 mg/kg/day × 17, at which point the catheter was removed and the patient sent home on weekly i.v. doses at 15 mg/kg. Toxicity, morbidity, and mortality were minimal with the intrahepatic arterial infusion treatment and the rigid criteria of improvement were met by 55% of the study cases. The survival rate of those patients who responded to the treatment was greater than the survival rate of those who failed to respond.

Chondrosarcomas are the second most common primary malignant tumor of bone. Chemotherapy for conventional chondrosarcomas is generally ineffective. Methylthioadenosine phosphorylase (MTAP) is a ubiquitous enzyme, essential in the salvage pathway of adenine and in methionine synthesis. MTAP-deficient cells are more susceptible than wild-type cells to pharmacologic inhibitors of de novo purine synthesis. Homozygous deletions of MTAP have been reported in hematologic and solid tumor malignancies. Based on these observations, we investigated the frequency of MTAP deletions in conventional, grade II chondrosarcomas by fluorescence in situ hybridization (FISH) analysis: 23 conventional, grade II chondrosarcoma patient samples from the Cleveland Clinic Foundation were analyzed for MTAP deletions. Nuclei were successfully extracted from 14 of 23 samples (61% evaluable) for FISH analysis: 7 of 14 samples (50%) showed either homozygous or hemizygous deletion of the MTAP gene, 6 of 14 (43%) failed to show deletion, and 1 of 14 (7%) was inconclusive. These findings suggest that approximately one-half of conventional, grade II chondrosarcomas may be preferentially sensitive to pharmacologic inhibitors of de novo purine synthesis. The present study led to development by the Intergroup Coalition Against Sarcomas of a phase II trial of pemetrexed, a multitargeted anti-folate, for advanced chondrosarcomas.

IL-2 therapy results in 10–20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mφ). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mφ (4-fold), whereas IL-2 alone had little effect. Mφ increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mφ expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-γ T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-γ capable of activating tumoricidal Mφ. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-γ production and expansion/activation of Mφ. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.

Interferons (IFNs) remain the most broadly active cytokines for cancer treatment, yet ones for which the full potential is not reached. IFNs have impacted positively on both quality and quantity of...

In general, the survival of patients with metastatic uveal melanoma is poor with a median survival of less than 6 months. Despite recent advances in management of uveal melanoma, the relative survival rate appears to have remained unchanged over the last 25 years. In this chapter, the pathogenesis, clinical features, and treatment of metastatic uveal melanoma are outlined.

The authors have noted anecdotal cases of extrauterine leiomyosarcomas (LMS) with estrogen receptor (ER) and progester-one receptor (PR) immunoreactivity. However, there are few studies that have compared ER and PR immunoexpression in LMS of uterine and extrauterine origin. The authors obtained a representative formalin-fixed, paraffin-embedded tissue block from cases of uterine LMS (n = 15) and extrauterine LMS (n = 16) from the archives of the Cleveland Clinic Foundation and performed immunohistochemical staining for ER and PR. Staining was evaluated by 2 observers in a semiquantitative manner using the following scale: 0, no nuclear staining; 1+, 1 to 25% of nuclei stained; 2+, 26 to 50% of nuclei stained; 3+, 51 to 75% of nuclei stained; 4+, 76 to 100% of nuclei stained. The majority of uterine LMS stained for ER (13 of 15, 87%), PR (12 of 15, 80%), or both ER and PR (12 of 15, 80%), with most cases showing 3+ or 4+ positive staining. For the extrauterine LMS cases, staining for ER was seen in 4 of 16 cases (25%), staining for PR was observed in 2 of 16 cases (13%), and staining for both ER and PR was seen in 2 of 16 cases (13%). One extrauterine LMS showed 4+ coexpression of ER and PR, but the remaining extrauterine cases showed only 1+ ER and/or PR immunoreactivity. These data suggest that most uterine LMS coexpress ER and PR, and most extrauterine LMS do not stain for these antigens. However, a subset of extrauterine LMS are ER and/or PR immunoreactive. This raises the possibility that hormonal manipulation may be beneficial in the treatment of these therapeutically recalcitrant tumors.

Objective: The protocol was designed to examine the biological effects and clinical activity of interferon-β in patients with platinum/taxane-resistant ovarian cancer. Methods: Patients with resistant ovarian and fallopian tube cancers and primary peritoneal carcinoma were treated with recombinant human interferon-β (Rebif®, Serono International) at doses ranging from 6 to 24 million international units (MIU)/day, based on their tolerance to therapy. Levels of IP-10, an interferon-inducible protein, were measured in the serum to evaluate the biological effects of the drug. Also, the peripheral blood mononuclear cells and serum were examined for the induction of previously described novel regulators of interferon-induced death. Results: Eighteen patients were treated, of whom 9 (50%) could be treated at the highest dose level (24 MIU). The major toxicities were fever, chills and fatigue. The median duration of therapy was 6 weeks (range 1–22). No objective responses were observed. IP-10 levels were significantly increased, compared with baseline, at 2, 4, and 6 weeks after initiation of therapy (p < 0.01). Conclusions: Recombinant human interferon-β produced a definite biological effect in the serum of treated patients, but this outcome was not translated into any clinically observable or meaningful impact on the disease process.

Interferon (IFN)-dependent cellular effects are mediated by transcriptional induction of responsive genes, collectively referred to as IFN-stimulated genes (ISGs). Which ISGs regulate the potent antiviral, antiproliferative, apoptosis-inducing, antiangiogenic, and immunologic effects of IFNs remains largely undetermined. To identify genes that might be useful for predicting or targeting apoptosis induction in response to IFNs, WM9 melanoma cells were assessed. WM9 cells had equivalent antiviral activity in response to IFN-beta and IFN-alpha2 but underwent apoptosis only in response to IFN-beta. RNA samples from WM9 cells and WM35 cells, a second melanoma cell line, treated with IFN-alpha2 or IFN-beta were assessed on oligonucleotide arrays. For 95% of genes assessed, IFN-beta was more potent than IFN-alpha2 in inducing ISG expression. Using a 22,000-gene oligonucleotide array, the largest yet reported for assessing ISG induction, approximately 910 genes were identified as induced by IFN-beta at 500 U/ml, and 260 ISGs were identified as significantly induced by IFN-beta at both 50 and 500 U/ml. Of these 260, 209 were defined as new ISGs based on the array analysis. Confirmation by Northern blot or semiquantitative or quantitative PCR was undertaken for 28, and all were confirmed. Nearly half of the 260 genes were functionally categorized as encoding growth-regulatory proteins. Of the 104 with described growth-regulatory function, 71 were induced more than three times by 500 U/ml and twice by 50 U/ml IFN-beta, and 48 of these were new ISGs. Included in this latter category were tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), XIAP-associated factor 1 (XAF1), galectin 9, a cyclin E binding protein, amphiphysin 1, MyD88, and several ubiquitin pathway genes. The diversity of stimulated genes suggests the full therapeutic potential of IFN regulation of gene expression has yet to be realized.

This study was designed to evaluate the toxicity and efficacy of cisplatin and WR-2721 in contrast to cisplatin alone for the therapy of measurable metastatic melanoma. Ninety-four patients with metastatic melanoma were randomized to receive either cisplatin at a dose of 150 mg/m2 and WR-2721 at a dose of 910 mg/m2, or cisplatin alone at a dose of 120 mg/m2. WR-2721 did not mitigate the toxic effects of cisplatin, and toxicity was increased in the WR-2721 plus cisplatin arm compared with cisplatin alone. For patients receiving cisplatin alone, the response rate was 16.3%; for those receiving cisplatin plus WR-2721, the response rate was 23.3%. The duration of response was 7.3 months. Median survival in the intent-to-treat analysis was 7.58 months. The study was terminated after accrual of 94 patients, with inadequate power to define an effect of WR-2721 on the duration of response and survival. In conclusion, cisplatin with WR-2721 showed an improved response rate over cisplatin alone. The lack of improved duration of response or impact on survival may be the result of the limited improvement of efficacy with the higher dosage of cisplatin in conjunction with WR-2721, or the limited number of patients accrued to this study. These factors, coupled with the failure of the combination to diminish toxicity, dampen enthusiasm for this combination.

ISG15 is a ubiquitin-like protein that conjugates to numerous proteins in cells treated with interferon or lipopolysaccharide. Dysregulation of protein ISG15 modification (ISGylation) in mice leads to decreased life expectancy, brain cell injury, and hypersensitivity to interferon. Although ISG15 was identified more than two decades ago, the exact biochemical and physiological functions of ISG15-modification remain unknown, and the proteins targeted by ISG15 have not been identified. The major purpose of this work was to identify ISG15 targets among well characterized proteins that could be used as models for biological studies. We purified ISGylated proteins from human thymus by immunoaffinity chromatography and analyzed ISG15 conjugates by a high-throughput Western blot screen (PowerBlotTM). We found that three key regulators of signal transduction, phospholipase Cγ1, Jak1, and ERK1 are modified by ISG15. In addition to that, we demonstrate that transcription factor Stat1, an immediate substrate of Jak1 kinase, is also ISGylated. Using whole cell protein extracts and phospholipase Cγ1 as an example we demonstrate that ISG15 conjugates are not accumulated in cells treated with specific inhibitors of proteasomes. Our work suggests a role for ISG15 in the regulation of multiple signal transduction pathways and offers attractive models to further elucidate the biochemical function of ISGylation.

Recombinant human interferon (IFN)-alpha was the first biotherapeutic agent approved by the US Food and Drug Administration for the treatment of a human malignancy. Its efficacy has also been demonstrated for treatment of several viral diseases. The human genome codes for 12 IFN-alpha proteins, with IFN alpha-1 and IFN alpha-2 accounting for the majority of the naturally occurring IFN-as. However, only subspecies of IFN alpha-2, recombinant human IFN alpha-2a and IFN alpha-2b, are commercially available in the United States. Other species of IFN-a may demonstrate equivalent or improved efficacy and have more tolerable side effects. This article describes ongoing preclinical and clinical studies of several new and modified IFN-alphas. A current phase I trial of a human recombinant IFN alpha-1 is described. Basic pharmacokinetics and clinical studies of polyethylene glycol (PEG) IFN alpha-2b are reviewed as well. Lastly, two novel types of IFN-a, one gene shuffled and one hybridized with human albumin, are summarized.

IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF-alpha related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-alpha, IFN-beta and IFN-gamma induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.

Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-α synergized to overcome IFN-α resistance in various human cancer cell lines in culture and eradicated IFN-α-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-α-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

Importance After identification of activating mutations of the KIT gene in gastrointestinal stromal tumor (GIST)—the most common sarcomaof the gastrointestinal tract—a phase 2 study demonstrated efficacy of imatinib mesylate in patients with metastatic GIST harboring a KIT exon 11 mutation. Initial results of long-term follow-up have found a survival benefit in this subgroup of patients. Obective To assess the long-term survival of patients with GIST who were treated in SWOG study S0033 and to present new molecular data regarding treatment outcomes. Design, Setting, and Participants In this follow-up of randomized clinical trial participants (from December 15, 2000, to September 1, 2001), patients were required to have advanced GIST that was not surgically curable. Postprotocol data collection occurred from August 29, 2011, to July 15, 2015. Using modern sequencing technologies, 20 cases originally classified as having wild-type tumors underwent reanalysis. This intergroup study was coordinated by SWOG, a cooperative group member within the National Clinical Trials Network, with participation by member/affiliate institutions. This follow-up was not planned as part of the initial study. Interventions Patients were randomized to 1 of 2 dose levels of imatinib mesylate, including 400 mg once daily (400 mg/d) vs 400 mg twice daily (800 mg/d), and were treated until disease progression or unacceptable toxic effects of the drug occurred. Main Outcomes and Measures The primary end point was overall survival. Updated survival data were correlated with clinical and molecular factors, and patterns of postprotocol therapies were enumerated and described in long-term survivors. Results Of 695 eligible patients (376 men [54.1%]; 319 women [45.9%]; mean [SD] age, 60.1 [14.0] years), 189 survived 8 years or longer, including 95 in the 400-mg/d dose arm and 94 in the 800-mg/d arm. The 10-year estimate of overall survival was 23% (95% CI, 20%-26%). Among 142 long-term survivors, imatinib was the sole therapy administered in 69 (48.6%), with additional systemic agents administered to 54 patients (38.0%). Resequencing studies of 20 cases originally classified as KIT/PDGFRA wild-type GIST revealed that 17 (85.0%) harbored a pathogenic mutation, most commonly a mutation of a subunit of the succinate dehydrogenase complex. Conclusions and Relevance A subset of patients with metastatic GIST experiences durable, long-term overall survival with imatinib treatment. Although this study provides guidance for management of GIST harboring the most common KIT and PDGFRA mutations, optimal management of other genotypic subtypes remains unclear. Trial Registration clinicaltrials.gov Identifier:NCT00009906

Importance This study helps to elucidate the appropriate surgical treatment for sinonasal melanoma. Objective To compare open resection (OR) and endoscopic resection (ER) as surgical approaches to sinonasal mucosal melanoma (SNM)and evaluate their associations with treatment-related outcomes. Design, Setting, and Participants Retrospective review of the medical records of 25 patients with sinonasal mucosal melanoma (SNM) treated by either OR or ER in an academic tertiary care medical center. Interventions The patients underwent either OR or ER of their SNM tumors. Main Outcomes and Measures Overall survival was the primary outcome measured; secondary outcomes were postoperative complications, lengths of hospital stay, patterns of failure, and disease-free survival. Results Thirteen patients with SNM underwent an OR, while 12 had ER of their tumors. The OR and ER groups did not differ significantly in demographic and tumor characteristics. In the OR vs ER group comparisons, mean age (67.8 vs 65.5 years) ( P = .63), the proportions of patients who received adjuvant radiotherapy (85% [n = 11] vs 92% [n = 11]) ( P > .99), and the proportion who achieved negative surgical margins on resection (54% [n = 7] vs 58% [n = 7]) ( P = .82) were similar. Overall all median survival (12.7 and 1.9 years) ( P = .87) and disease-free survival (1.9 and 1.2 years) ( P = .72) were modest and did not differ between OR and ER groups, respectively. Likewise, the OR and ER groups, respectively, showed comparable mean lengths of hospital stay (3.6 and 3.8 days) ( P = .87), rates of postoperative bleeding (8% [n = 1] and 17% [n = 2]) ( P = .59), and rates of cerebrospinal fluid leak (15% [n = 2] and 25% [n = 3]) ( P = .64). In addition, the OR and ER groups, respectively, had high rates of local (23% [n = 3] and 8% [n = 1]) ( P = .59), distant (15% [n = 2] and 25% [n = 3]) ( P = .64), and multiple failures (15% [n = 2] and 25% [n = 3]) ( P = .64). Conclusions and Relevance This retrospective study of a rare disease suggests that endoscopic resection of sinonasal melanoma offers an attractive, minimally invasive surgical option. In the hands of an experienced surgeon, an endoscopic approach yields survival and morbidity outcomes comparable to those of an open approach.

Our prior phase I study of the combination of vascular endothelial growth factor (VEGF) antibody, bevacizumab, and VEGF receptor (VEGFR) inhibitor, sunitinib, in advanced solid tumors identified an encouraging response evaluation. An expansion phase of this study was thus undertaken to obtain further safety data, response assessment and characterization of pharmacodynamic biomarkers in melanoma, renal, and adrenal carcinoma patients. Patients with metastatic solid tumors received sunitinib (37.5 mg/d, 4 wk on/2 wk off) and bevacizumab (5 mg/kg intravenously every 2 wk). Responses were assessed every 2 cycles. Serum levels of angiogenic molecules were measured using ELISA assays. Twenty-two patients were enrolled, including 11 melanoma, 5 renal cell carcinoma (RCC), 5 adrenal cancer, and 1 angiosarcoma. Grade 3 or higher adverse events were observed in 15 patients, including hypertension (41%), thrombocytopenia (23%), and fatigue (14%). Three RCC patients, and 1 melanoma patient developed thrombotic microangiopathy (TMA). Partial response (PR) occurred in 21% patients, including melanoma (2), adrenal (1), and renal (1) carcinomas. Overall, 6 patients demonstrated some reduction in their tumor burden. Serum VEGF and several other proangiogenic proteins declined over the first 4 wk of treatment whereas the putative VEGF-resistant protein, prokineticin-2, increased over 10-fold. Occurrence of TMA related to dual VEGF/VEGFR inhibition can result from systemic or nephron specific injury even in non-renal malignancies. While the combination of sunitinib and bevacizumab was clinically efficacious in renal cell carcinoma and melanoma, the observance of microangiopathy, even in non-RCC patients, is a significant toxicity that precludes further clinical development.

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME ) targeting the Flt-1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo-controlled, single-dose escalation study, ribozyme was administered as a 4-hour IV infusion of 10 or 30 mg/m 2 or as a SC bolus of 20 mg/m 2 . Peak ribozyme plasma concentrations of 1.5 and 3.8 pg/mL were observed after the 10 and 30 mg/m 2 IV infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m 2 . Peak concentrations of 0.9 μg/mL were observed approximately 3.25 hours after a 20 mg/m 2 SC bolus of ribozyme. The dose-normalized A UCs obtained after SC dosing were compared to the mean dose-normalized AUC after IV dosing to estimate an absolute SC bioavailability (f) of approximately 69%. An average elimination half-life of 28 to 40 minutes was observed after IV administration, which increased to 209 minutes after SC administration, Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4-hour IV infusion of up to 30 mg/m 2 or a single SC bolus of 20 mg/m 2 .

IFNs were the first new therapeutic products resulting from recombinant DNA technology. IFNs were also the first human proteins effective in cancer treatment. There is however much to be discovered which will lead to new clinical applications. Areas which represent major research challenges for full understanding and application of the IFN system are: (i) the diversity of the IFN family; (ii) the role of induction; (iii) molecular mechanism of action; (iv) cellular modulatory effects; (v) advantages of combinations, and (vi) identification of new therapeutic indications. This review will emphasize the diversity of the IFN family and chemical modifications which will result in second-generation IFNs. Pre-clinical and clinical findings form the basis for new therapeutic directions in chronic myelogenous leukemia, lymphomas, myelomas, melanoma, urologic malignancies, primary brain tumors, and ovarian carcinoma.