Paul E.G. Kristjansen

Clinical and biological efficacy of recombinant human interleukin-21 in patients with stage IV malignant melanoma without prior treatment : a phase IIa trial

Purpose: Human interleukin-21 (IL-21) is a class I cytokine that mediates activation of CD8+ T cells, natural killer (NK) cells, and other cell types. We report final clinical and biological results of a phase II study of recombinant human IL-21 (rIL-21) in patients with metastatic melanoma. Experimental Design: Open-label, single-arm, two-stage trial. Eligibility criteria: unresectable metastatic melanoma, measurable disease by Response Evaluation Criteria in Solid Tumors, no prior systemic therapy (adjuvant IFN permitted), adequate major organ function, good performance status, no significant autoimmune disease, and life expectancy at least 4 months. Primary objective: antitumor efficacy (response rate). Secondary objectives: safety, blood biomarkers, and generation of anti-rIL-21 antibodies. rIL-21 (30 μg/kg/dose) was administered by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment). Results: Stage I of the study comprised 14 patients. One confirmed complete response (CR) was observed, and as per protocol, 10 more patients were accrued to stage II (total n = 24: 10 female and 14 male). Best tumor response included one confirmed CR and one confirmed partial response, both with lung metastases. Treatment was overall well tolerated. Biomarker analyses showed increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells. Conclusions: rIL-21 administered at 30 μg/kg/d in 5-day cycles every second week is biologically active and well tolerated in patients with metastatic melanoma. Confirmed responses, including one CR, were observed.

Regulation of YKL-40 expression during genotoxic or microenvironmental stress in human glioblastoma cells

YKL‐40 is a 40 kDa secreted glycoprotein belonging to the family of ‘mammalian chitinase‐like proteins’, but without chitinase activity. YKL‐40 has a proliferative effect on fibroblasts, chondrocytes and synoviocytes, and chemotactic effect on endothelium and vascular smooth muscle cells. Elevated YKL‐40 levels are found in serum of patients with diseases characterized by inflammation, fibrosis and tissue remodeling. Several studies have reported that high serum YKL‐40 levels in patients with cancer are associated with poor prognosis. YKL‐40 expression is strongly elevated in serum and biopsy material from glioblastomas patients. We investigated the expression of YKL‐40 in three human malignant glioma cell lines exposed to different types of stress. Whereas a polymerase chain reaction transcript was detectable in all three cell lines, only U87 produced measurable amounts of YKL‐40 protein. In U87, hypoxia and ionizing radiation induced a significant increase in YKL‐40 after 24–48 h. The hypoxic induction of YKL‐40 was independent of HIF1. Etoposide, ceramide, serum depletion and confluence all led to elevated YKL‐40. Inhibition of p53 augmented the YKL‐40 expression indicating that YKL‐40 is attenuated by p53. In contrast, both basic fibroblast growth factor and tumor necrosing factor‐α repressed YKL‐40. These are the first data on regulation of YKL‐40 in cancer cells. Diverse types of stress resulted in YKL‐40 elevation, which strongly supports an involvement of YKL‐40 in the malignant phenotype as a cellular survival factor in an adverse microenvironment. (Cancer Sci 2005; 96: 183–190)

Aerobic glycolysis in cancers: Implications for the usability of oxygen‐responsive genes and fluorodeoxyglucose‐PET as markers of tissue hypoxia

The hypoxia‐responsiveness of the glycolytic machinery may allow pretreatment identification of hypoxic tumors from HIF‐1 targets (e.g., Glut‐1) or [18F]‐fluorodeoxyglucose positron emission tomography but results have been mixed. We hypothesized that this discrepancy is an inevitable consequence of elevated aerobic glycolysis in tumors (Warburg effect) as energetics in predominantly glycolytic cells is little affected by hypoxia. Accordingly, we characterized glycolytic and mitochondrial ATP generation in normoxic and anoxic cell lines. Measurements demonstrated that most cancer cells rely largely on aerobic glycolysis as it accounts for 56–63% of their ATP budget, but in the cervical carcinoma SiHa, ATP synthesis was mainly mitochondrial. Moreover, the stimulatory effect of anoxia on glycolytic flux was inversely correlated to the relative reliance on aerobic glycolysis. Next, tumor cells representing a Warburg or a nonglycolytic phenotype were grown in mice and spatial patterns of hypoxia (pimonidazole‐stained), Glut‐1 expression and 18F‐FDG uptake were analysed on sectioned tumors. Only in SiHa tumors did foci of elevated glucose metabolism consistently colocalize with regions of hypoxia and elevated Glut‐1 expression. In contrast, spatial patterns of Glut‐1 and pimonidazole staining correlated reasonably well in all tumors. In conclusion, Glut‐1s value as a hypoxia marker is not severely restricted by aerobic glycolysis. In contrast, the specificity of 18F‐FDG uptake and Glut‐1 expression as markers of regional hypoxia and glucose metabolism, respectively, scales inversely with the intensity of the Warburg effect. This linkage suggests that multi‐tracer imaging combining FDG and hypoxia‐specific markers may provide therapeutically relevant information on tumor energetic phenotypes.

Tumor angiogenesis ‐ a new therapeutic target in gliomas

Tumor growth is critically dependent on angiogenesis, which is sprouting of new vessels from pre‐existing vasculature. This process is regulated by inducers and inhibitors released from tumor cells, endothelial cells, and macrophages. Brain tumors, especially glioblastoma multiforme, have significant angiogenic activity primarily by the expression of the angiogenic factor VEGE Anti‐angiogenic therapy represents a new promising therapeutic modality in solid tumors. Several agents are currently under evaluation in clinical trials. The present review describes the principal inducers and inhibitors of angiogenesis in tumors and summarizes what is known about their mechanisms of action in relation to CNS tumors. Potential areas for clinical use are also discussed.

Interleukin-21 Signaling: Functions in Cancer and Autoimmunity

Interleukin-21 (IL-21) is a cytokine with structural and sequence homology to IL-2 and IL-15, yet possesses several biological properties distinct from these cytokines. IL-21 is produced mainly by activated CD4+ T cells and natural killer T cells and mediates its activity by binding to the IL-21 receptor (IL-21R), consisting of an IL-21–specific α chain (IL-21Rα; JAK/STAT) that heterodimerizes with the common γ chain (CD132). Intracellular signaling occurs through the Janus-activated kinase/signal transducer and activator of transcription pathways. Physiologic expression of IL-21R is restricted to lymphoid tissues and peripheral blood mononuclear cells; however, other tissues such as epithelium, synovium, or transformed cells can acquire expression of both components of IL-21R heterodimer. IL-21 has complex activities on a wide variety of cell types, leading to enhancement of adaptive T-cell immunity, antibody production, activation of natural killer cell subtypes, and opposition to suppressive effects mediated by regulatory T cells. Functionally, these activities promote immune responses and point to a physiologic role of IL-21 in autoimmunity and immune enhancement. Therapeutic manipulation of IL-21 activity may allow improved immunotherapy for cancer as well as insights into autoimmune disease. Recently conducted phase 1 trials in metastatic melanoma and renal cell carcinoma have shown that recombinant IL-21 has a favorable safety profile and support its continued investigation as a potential anticancer drug.

How few cancer cells can be detected by positron emission tomography? A frequent question addressed by an in vitro study

PurposePositron emission tomography (PET) has gained widespread use in cancer diagnosis and treatment, but how many malignant cells are required for a tumour to be detected by PET?MethodsThree human cancer cell lines [glioblastoma and two subtypes of small cell lung cancer (SCLC)] in concentrations from 104 to 107 were seeded on six-well plates or plastic tubes and treated with [18F]fluorodeoxy-glucose (FDG) in vitro. FDG retention was measured in a PET/CT scanner and in a calibrated well counter. The clinical situation was simulated using a cylinder phantom with a background concentration of FDG.ResultsThe theoretical detection limit was found to be around 105 malignant cells. In a cylinder phantom the detection limit was increased by a factor of 10. The FDG retention by the glioblastoma cell line was significantly higher than the activity of the SCLC cell line. FDG retention measured by PET and a gamma counter was closely correlated to the number of cells and a linear relationship was found.DiscussionThe detection limit of PET is in the magnitude of 105 to 106 malignant cells. The experimental set-up was robust and well suited as a platform for further investigations of factors influencing the detection limit of PET.

Differential regulation of VEGF, HIF1α and angiopoietin‐1, ‐2 and ‐4 by hypoxia and ionizing radiation in human glioblastoma

We examined how ionizing radiation (IR) delivered under either severe hypoxia (< 0.1% O2) or normoxia affects the expression of hypoxia inducible factor 1α (HIF‐1α) and the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins 1, 2 and 4 in U87 human glioblastoma cells. IR was delivered as single doses of 0, 2, 5, 10 and 20 Gy after 6‐hr hypoxic incubation and in normoxic controls. Irradiation at any dose did not affect the cellular protein levels of any of the angiopoietins, whereas hypoxia led to increasing levels of both angiopoietin‐4 and angiopoietin‐2. Levels of angiopoietin‐1 protein were unaltered throughout the observation period. A dose‐dependent increase in levels of secreted VEGF in the medium occurred after IR at doses from 5–20 Gy. In hypoxic cells, 20 Gy IR induced an additional significant increase in VEGF relative to nonirradiated hypoxic control cells with elevated baseline VEGF levels induced by hypoxia. HIF‐1α and glucose transporter‐1 (Glut‐1) were not correspondingly upregulated by IR. Blocking HIF‐1α by antisense treatment induced a reduced baseline VEGF at normoxia, while the relative upregulation of VEGF by IR was unaffected. These data provide evidence that VEGF is upregulated by IR by mechanisms independent of HIF‐1 transactivation.

The role of prophylactic cranial irradiation in the management of small cell lung cancer

Those patients who benefit most in terms of increased survival from the current treatment of small cell lung cancer (SCLC) are also those who run the greatest risk of developing brain metastases. In a compilation of 11 studies with 1688 patients, Pedersen (1) calculated that about 10% had central nervous system (CNS) metastases at diagnosis of SCLC, while a further 20% (range 7-30%) developed CNS metastases during therapy. At autopsy the frequency was found to be about 50% (1, 2). An increase in frequency of brain metastases in SCLC with increased survival was predicted as early as 1973 (3), and the prediction was confirmed in 1979 by Nugent et al. (4), who demonstrated an actuarial probability of developing brain metastases of 80% 2 years after diagnosis. Similar tendencies were subsequently demonstrated by others (5), and in a series from the Finsen Institute in Copenhagen the cumulative risk of brain metastases reached 50% at 2 years survival (1). An early and widely disseminated hypothesis explaining the increase in CNS metastases in patients with prolonged survival describe the brain as a pharmacologic sanctuary, where metastases can grow shielded from cytostatic agents by the blood-brain-barrier (BBB). This hypothesis has to some degree been corroborated by the clinical experience with childhood leukemia, where prophylactic cranial irradiation (PCI) is an integrated part of most treatment programmes. When CNS metastases are diagnosed by intravenous iodine contrast-enhanced CT-scan, the BBB is not intact in the tumor areas, and such areas therefore should be accessible to drugs according to the same principles that govern extracranial metastases. During the last decade, several reports about the effect of systemic chemotherapy on brain metastases from SCLC have been published, see (6, 7) for a review. Thus, the concept of the brain as a pharmacologic sanctuary site for metastases is inconsistent with newer clinical observations. In spite of the fact that clinical brain metastasis is an ominous prognostic indicator, it is disputable what influence the presence of brain metastases per se, if actively

Biochemical and physiological changes induced by nicotinamide in a C3H mouse mammary carcinoma and CDF1 mice

We have continued our investigation into the mechanism by which nicotinamide can enhance radiation damage in tumors, using a C3H mouse mammary carcinoma grown in CDF1 mice. Biochemical analysis of tumor extracts showed that nicotinamide (1000 mg/kg; i.p.) increased the ATP/Pi and ATP/ADP + AMP ratios. This change in metabolic activity was consistent with nicotinamide increasing tumor oxygenation. Moreover, the greatest effect occurred 0.5-2.5 hr after drug injection, a time at which radiosensitization by nicotinamide in this tumor had previously been shown to be maximal. These changes were observed without any apparent modification in tumor blood perfusion, measured using the 86-RbCl uptake procedure, and occurred despite nicotinamide producing a 50% decrease in mean arterial blood pressure, estimated directly by a carotid cannulation technique.

Hypoxia-induced secretion of macrophage migration-inhibitory factor from MCF-7 breast cancer cells is regulated in a hypoxia-inducible factor-independent manner.

The cytokine MIF is over-expressed in tumors and is associated with tumor proliferation, angiogenesis and metastasis. Hypoxia, a hallmark feature of tumors, increases MIF expression from tumor cells. We examined the role of hypoxia-inducible transcription factors on MIF secretion from MCF-7 breast carcinoma cells. Secretion of MIF was induced by hypoxia after 24h but up-regulation of MIF mRNA was minimal. Inhibition of HIF-1alpha, HIF-2alpha, NF-kappaB and C/EBPbeta using siRNA had no effect on hypoxia-induced MIF secretion. However, inhibition of transcription and translation significantly decreased MIF production, suggesting that hypoxia-induced secretion of MIF in MCF-7 cells is via an alternative pathway.